Urotensin II-related peptides, Urp1 and Urp2, control zebrafish spine morphology

  1. Elizabeth A Bearce
  2. Zoe H Irons
  3. Johnathan R O'Hara-Smith
  4. Colin J Kuhns
  5. Sophie I Fisher
  6. William E Crow
  7. Daniel T Grimes

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Abstract

The spine provides structure and support to the body, yet how it develops its characteristic morphology as the organism grows is little understood. This is underscored by the commonality of conditions in which the spine curves abnormally such as scoliosis, kyphosis, and lordosis. Understanding the origin of these spinal curves has been challenging in part due to the lack of appropriate animal models. Recently, zebrafish have emerged as promising tools with which to understand the origin of spinal curves. Using zebrafish, we demonstrate that the urotensin II-related peptides (URPs), Urp1 and Urp2, are essential for maintaining spine morphology. Urp1 and Urp2 are 10-amino acid cyclic peptides expressed by neurons lining the central canal of the spinal cord. Upon combined genetic loss of Urp1 and Urp2, adolescent-onset planar curves manifested in the caudal region of the spine. Highly similar curves were caused by mutation of Uts2r3, an URP receptor. Quantitative comparisons revealed that urotensin-associated curves were distinct from other zebrafish spinal curve mutants in curve position and direction. Last, we found that the Reissner fiber, a proteinaceous thread that sits in the central canal and has been implicated in the control of spine morphology, breaks down prior to curve formation in mutants with perturbed cilia motility but was unaffected by loss of Uts2r3. This suggests a Reissner fiber-independent mechanism of curvature in urotensin-deficient mutants. Overall, our results show that Urp1 and Urp2 control zebrafish spine morphology and establish new animal models of spine deformity.

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  1. Version published to 10.7554/elife.83883 on eLife
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    Reply to the reviewers

    1. General Statements [optional]

    See cover letter for more details.

    Summary of response to reviewers:

    We were immensely pleased that the reviewers considered our conclusions “well supported” and our study “beautifully executed”. Reviewers also recognized the significance of our work. Reviewer 1 stated that “building a model that describes one of these pathways will allow us to begin to test therapies to treat or prevent scoliosis” then noted that we “help to build a larger model of normal spine morphogenesis” and that this is “important”. Reviewer 2 called our work an “exciting advance in our understanding of one of the essential signaling pathways that help regulate body axis straightening and spine morphogenesis in zebrafish” and mentioned that our work “may also help to further our understanding of the etiology and pathophysiology of multiple forms of neuromuscular scoliosis in humans”. Reviewer 3 agreed, stating that our work “adds important information on the role of urotensin signaling in spine formation” and noted that it is timely: “findings are of special significance in the light of recent reports that mutations in UTS2R3 show association with spinal curvature in patients with adolescent idiopathic scoliosis”.

    We thank the three reviewers for reading our research and providing feedback. In all cases, we have incorporated (or plan to incorporate) their suggestions, and we believe this has (will) make our manuscript much stronger. Indeed, reviewers had only a small number of “major points”, and all are easily addressed as summarized below. We have already addressed some of those “major points”, as well as the majority of “minor points” raised by reviewers, in our current draft. We expect that all comments can be fully addressed within around 1 month.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are plannedto address the points raised by the referees.

    We have divided our responses by whether the reviewers considered their points major or minor. All points have already been, or will soon be, fully addressed.

    Major points

    Reviewer 1

    The key conclusions are well supported, see below for my two major issues.

    Please don't call this lordosis. Lordosis or hyperlordosis effects lumbar vertebra. The curve in the lumbar region shifts body weight so that human gait is more efficient that that in the great apes, or so the story goes. Zebrafish do not have lumbar vertebra equivalents or a natural curve in the caudal region. Similarly, fish do not have the equivalent vertebra to generate kyphosis, which is again a hyper flexion of a normal human spinal curve. Instead zebrafish have Weberian, precaudal and caudal vertebra. It would be so much more useful for the field if the authors used these terms and specified ranges, i.e. numbered vertebrae, that are effected so we can directly and accurately compare regions of defects between zebrafish mutants. It would help to make the point that the uts2r3 mutant has more caudally located curves than urp1/2 double mutants. We appreciate this point and agree with the reviewer. Lordosis (or hyperlordosis) is indeed the accentuation of a curve which naturally exists in humans but not zebrafish. We called the phenotype of Urotensin pathway mutants ‘lordosis’ or ‘lordosis-like’ because of the position of the curves — in caudal vertebrae, which are evolutionarily and positionally equivalent to lumbar vertebrae, though they are structurally different to human lumbar vertebrae. To address this comment, we will no longer refer to the phenotype as lordosis in our Introduction or Results sections and we will expand our Discussion to include this point raised by the reviewer.

    1. The observation that urp1/2 double mutants have curves only in the D/V plane and almost completely lack side-to-side curves is noteworthy. Does the urp1-/-urp2-/- mutant uncouple two systems for posture? If this separate a DV from side-to-side postural control system, that would be very interesting. It is particularly important to describe how penetrant the phenotype is and how many times it was observed. See 9 minor comments. It would help the reader if the authors explicitly described the features that they see in the cfap298 mutant that constitute lateral curves and that are lacking in urp1/2 (e.g. in figure 4E).

    We plan to expand the figure and analysis describing D/V curves and M/L curves. While our first draft included only cfap298 and urp1-∆P;urp2-∆P mutants, our next draft will also incorporate uts2r3 and pkd2l1 mutants. We have already scanned cohorts of all mutant fish, and so the remaining work to render and quantify the degree of lateral curvature will not take long. This will allow us to conclusively determine whether these different mutations indeed uncouple two systems controlling posture in different directions. As the reviewer requests, we will include all fish analyzed in either main or supplementary figures, include numbers in figure legends, and quantify the penetrance of M/L and D/V curves.

    We have also generated cfap298;urp1-∆P;urp2-∆P triple mutants and are currently scanning them to reveal skeletal form. Preliminary data suggests triple mutants have three-dimensional curves but D/V curves are more severe in triple mutants than in cfap298 mutants alone. This makes sense if Urp1/Urp2 are important for controlling D/V spinal shape and, as our qPCR shows, Urp1/Urp2 are downregulated but not lost completely in cfap298 mutants. It also furthers the notion that cilia motility controls D/V and M/L curves by separable mechanisms. * *

    Reviewer 2

    Need to show that the CRISPANT targeting was effective for mutagenesis at each loci screened in the work presented in Figure 1E. In Figure 1E, we presented the phenotypes of crispant embryos (i.e. embryos injected with four gRNAs targeting a specific gene alongside relatively high doses of Cas9 protein; see schematic in Figure 1G). In positive controls (cfap298 and sspo), crispants showed the expected phenotype in all cases (Figure 1E and see Figure 1H for quantitation). As for germline mutants, urp1 and urp2 crispants showed no early axial phenotypes (Figure 1E and 1H). As such, the reviewer requests that we perform molecular assays to determine whether mutagenesis was successful in these embryos. To do so, we will perform either T7 assays or next-generation/Sanger sequencing of mutated loci. This will allow us to determine and quantify the effectiveness of our mutagenesis. Results will be shared in a new supplementary figure. These assays are straightforward and we expect they will not take very long to complete. Indeed, we have performed these assays previously for other genes (e.g. Grimes et al., 2019 and several unpublished genes). We have achieved high levels of mutagenesis in all cases, making us very confident that we will achieve similarly high levels of mutagenesis in this case.

    Reviewer 3

    The addition of the F0 crispant experiment to show that the pro-peptide of urp1/2 does not have a function and is responsible for the difference between the observed morpholino and the crispr phenotype was important. However, since no phenotype was observed in crispants it is important to add evidence of induced cuts for all guide RNAs used in the crispant experiment. These control experiments might have been done already. If not, they can easily be done in a short period of time by performance of T7 assays on injected fish and would not require additional reagents. This is the same point raised by reviewer 2 and so we refer to the response above. In summary, we agree with the reviewer and we are currently performing these suggested experiments which are straightforward and working well.

    The authors claim that there were no structural defects observed in urp1/2 double mutants. However, the hemal arch in figure 3 E seems to be deformed. This could be normal variance or a phenotype. This can be addressed by simple reinspection of the scans.

    We believe there are no major vertebral structural defects that could be attributed to causing the spinal curves because vertebrae are well-formed in mutants and we see no defects in the initial patterning of vertebrae in our calcein experiments. However, since urp1-∆P;urp2-∆P and uts2r3 mutant spines are curved, the vertebrae are a little misshapen. We plan two revisions, one textual and one analytical.

    First, we will make clear in our textual edits that some vertebrae are slightly misshapen, as occurs in non-congenital forms of human spinal curve disease (in congenital forms, the shape defects are more striking and likely causative in the curvature). We agree with the reviewer that stating that there is a lack of vertebral structural defects lacked nuance, so we will expand on this in our next draft.

    Second, we will quantify vertebral shapes in spinal curve mutants and report these data in our next draft. After reinspection of the scans, as the reviewer suggested, we believe it would be informative for our readers to see quantitation of vertebral shape. We expect these data to more rigorously back up our statements about ‘minor structural differences’ of vertebrae between uncurved and curved individuals. We have already begun this work, and completing it should only take a few more weeks. As an example, we have measured the shape of centra by calculating aspect ratios in wild-type and urp1-∆P;urp2-∆P double mutants in curved regions of the spine:

    These preliminary data already make clear that there are indeed subtle morphological differences between vertebrae in mutants and wild-type, as occurs in human spinal curve deformities. We will present completed versions of these data (several parameters that describe vertebral shape) in our next draft and provide comments about whether such changes could be causative in spinal curve etiology as occurs in congenital-type scoliosis.

    Minor points

    Reviewer 1

    Supplementary FigS3B How to measure the Cobb Angle is unclear. Why is the first curve not counted? I count 3 curves. First a ventral displacement, then a dorsal to ventral return, then a sharp flex before the tail. How to measure Cobb angle might be easier to explain if the figure is expanded into steps. Identify the apical vertebra, then showing how the lines are drawn parallel to those vertebrae, then where the measured angle forms between the lines perpendicular to the drawn parallel lines.

    We will more thoroughly explain how Cobb angle is measured in our next draft.

    5a. I think we (zebrafish biologists) need be explicit about what we mean with "without vertebral defects." What do we count as defects? Vertebrae can be fused, bent, shortened or the growing edges can be slanted. In Figure 3E, and movie7, it is clear that the highlighted mutant vertebrae are shorter than WT. The growing ends of normal vertebra are perpendicular to the long axis of the vertebra. In the mutants the ends are slanted. Please define in the text what you consider a relevant vertebral defect, because these vertebrae have defects. Or are you only considering the calcein stained centra at 10dpf?

    We strongly agree with the reviewer. As described more thoroughly above in response to Major Comment – Reviewer 3, we plan both textual edits and new quantitation of vertebral shape to address this comment. Our quantitation indeed shows some vertebrae are shorter in mutants as the reviewer noticed. We also plan a new paragraph in the Discussion section which will speak about the issue of what zebrafish biologists might mean by “without vertebral defects”.

    5b. Do you want to base your patterning conclusion on primarily the calcein data as these are closer to the notochord patterning time window. Please anchor this conclusion to a specific time or standard length e.g. 10dpf/5.6mm.

    When we edit our descriptions of vertebral defects, and include new quantitative data on the shape of vertebrae, we will be clear that the vertebrae are slightly structurally malformed. In addition, when we speak of the calcein data, we will anchor those conclusions to the specific timepoint best studied by this method, as the reviewer suggests.

    "At 30 dpf... several mutants exhibited a significant curve in the pre-caudal vertebrae, in addition to a caudal curve (Fig. 3D and S3C). Since pre-caudal curves were rare in mutants at 3-months, this suggested that curve location is dynamic".The frequency of this observation is important. Does it effect all or a fraction of mutants? Can you provide some numbers to anchor these observations? Maybe fractions e.g.. 3 of 4 fish had precaudal curves at 30pdf, and 0 of 10 fish had precaudal curves by 3 mpf?

    In our next draft, we will provide numbers of fish examined at 30 dpf and also show graphical summaries of curve position (as we did for younger fish). Last, all scans will be included in a new supplementary figure.

    The description of the pkd2l1 mutant, instead of terming it kyphosis can you tell the reader the vertebra number at the peak of the curve. The authors say the pkd2l1 mutant is highly distinct from urp1/urp2-/-, but the reader needs to hear exactly what is distinct. For example, does this mutant have both lateral and D/V curves?

    We have now scanned several pkd2l1 mutant fish and we will include images of pkd2l1 mutants at two different timepoints together with quantitation of curve position. Our results agreed with those previously published for this mutant line (Sternberg et al., 2018) but we believe it is important for our readers to see side-by-side images and quantitation so they can see the distinctions.

    At 3-months of age, pkd2l1 mutants essentially appear wild-type but by around 12-months they have developed a D/V curve in the pre-caudal vertebrae. They do not exhibit M/L curves; we will quantify this and include these data in our Figure about M/L deviation.

    We called the phenotype displayed by pkd2l1 mutants “kyphosis” to be in line with a previous publication describing these mutants (Sternberg et al., 2018). We will add new wording in the Discussion about whether or not zebrafish can truly model kyphosis and lordosis (see response to Reviewer 1 major comment above), and we make clear in our Results that the phenotype has “been argued to model kyphosis (Sternberg et al., 2018)” rather than “is kyphosis”.

    It is intriguing that pkd2l1 mutants do not exhibit any curves until much later in life than urp1-∆P;urp2-∆P and uts2r3mutants. Inspired by this finding, we aged urp1-∆P and urp2-∆P single mutants and found that they go on to develop D/V curves by 12-months i.e.

    •                                *3-months                     12-months                               Position of curve

    *urp1-∆P *no curves mild D/V curves Mostly caudal

    urp2-∆P mild D/V curves intermediate D/V curves Mostly caudal

    urp1-∆P;urp2-∆P severe D/V curves severe D/V curves Mostly caudal

    uts2r3 severe D/V curves severe D/V curves Mostly caudal

    cfap298 severe 3D curves severe 3D curves Caudal and pre-caudal

    *pkd2l1 *no curves mild D/V curves Mostly pre-caudal

    Phenotypes in urp1-∆P and urp2-∆P single mutants upon aging shows: 1) Urp1 and Urp2 are not entirely redundant in long-term spine maintenance and 2) proper Urp1/Urp2 dose is essential. We will include these new data in our next draft.

    Does uts2r3-/- have no /minimal side-to-side curves like urp1/urp2-/-?

    This is an interesting question. To address it, we will add images of uts2r3 mutant spines from the dorsal aspect and include them with our new quantitation of lateral curvature. To sum, the reviewer’s suggestion is correct – there are minimal side-to-side curves in uts2r3 mutants.

    One finding that deserves more discussion is the observation that urp1/urp2 double mutants have almost no side-to-side defects and all the obvious bends are in the D/V plane. Does this uncouple two systems for posture? Please consider the following paper. It shows a proprioception system that maintains normal side-to-side posture. A spinal organ of proprioception for integrated motor action feedback. Picton LD, Bertuzzi M, Pallucchi I, Fontanel P, Dahlberg E, Björnfors ER, Iacoviello F, Shearing PR, El Manira A. Neuron. 2021 Apr 7;109(7):1188-1201.e7. doi: 10.1016/j.neuron.2021.01.018. Epub 2021 Feb 11. PMID: 33577748

    Thank you for pointing out this manuscript. We will include it in our expanded Discussion.

    Reviewer 2

    Fig 3F: might be improved by making the images black and white and possibly inverted. It is not easy to clearly see the vertebrae as is.

    Thanks for the suggestion, we will make this change.

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

    Minor points

    Reviewer 1

    Figure 1D legend says urp1 is expressed in dorsal while urp2 is express in all CSF-cNeurons, but the image for urp1 shows only ventral cells in WT, while the image for urp2 shows the same cells ...and more dorsal cells. Please replace image with one that matches the text. Apologies for this, we have now corrected it. The image was correct but we accidentally wrote “dorsal” instead of “ventral” when describing the CSF-cN sub-population harboring urp1 transcripts.

    In Figure 2H, the position of curve apex graphic, how many fish were examined? In 2f it looks like n=8 and n=9. Can this info be added to the figure?

    We have now included the number examined in the legend.

    I did not find legends for the movies. The first call to the movies calls movies 1-3 without explaining what each shows. The labels on the downloaded files are not informative.

    Apologies for forgetting to submit these. We have now added informative Movie legends.

    Reviewer 3

    It would be helpful to the reader to add a little more information on urp1 and upr2 proteins and their processing to make it clear while only the 3' region of the protein was targeted to induce mutations. We have incorporated textual edits to make this more clear. We now state in the second sentence of the Results section:

    Urp1 and Urp2 are encoded by 5-exon genes with the final exon coding for the 10-amino acid peptides that are released by cleavage from the pro-domain (Fig. 1A).

    Together with Fig. 1A and Supplementary Fig. 1, we hope it is now clear to readers how Urp1 and Urp2 are generated from a 5-exon gene encoding the pro-domain and the peptide, which are separated by cleavage.

    It would also be helpful to the reader to have a schematic indicating the guide target sites (they could be added to figure S1 C + D) in the protein to be able to interpret the result more easily.

    Done!

    Figure 5: Addition of a square to H would help understand were the pictures in D-F were taken.

    Done!

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

    N/A. We are performing all experiments/analyses requested by reviewers.

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    Referee #3

    Evidence, reproducibility and clarity

    The presented work by Bearce et al. is based on the hypothesis that urp1 and urp2, and their receptor uts2r3 play a role during zebrafish spine development. Previously it had been shown that cilia function as well as Reissner fiber formation are important for spine development and that both cilia motility and the Reissner fiber influence urp1/2 expression. Further, morpholino knock-down of upr1/2 did show the typical curly down phenotype observed in cilia and RF mutants. The authors generate CRISPR mutants for urp1, urp2 by targeting the 10-amino acid secreted peptides and do not find an early phenotype in single, double or maternal zygotic mutants or cripants. However, they observe a late onset curvature of the spine in urp1/2 double mutants and in generated uts2r3 single mutant. Spinal curvature was assessed through measurement of the Cobb angel in microCT scans and compared with other scoliosis mutants. This analysis revealed similarities between urp1/2 and uts2r3 mutants and differences with curvatures observed in cilia motility (cfap298) or Reissner fiber (sspo) mutants, which did show decreased expression levels of urp1 and 2. These differences in spine curvature do indicate that the phenotypes are not caused by the same mechanism. Analysis of the Reissner fiber in transgenic animals did show no defects.

    Major points:

    The paper is generally well written and easy to follow. All experiments are described in sufficient detail and reagents are listed. However, there are two points that should be addressed to strengthen the conclusion of the paper.

    1. The addition of the F0 crispant experiment to show that the pro-peptide of urp1/2 does not have a function and is responsible for the difference between the observed morpholino and the crispr phenotype was important. However, since no phenotype was observed in crispants it is important to add evidence of induced cuts for all guide RNAs used in the crispant experiment. These control experiments might have been done already. If not, they can easily be done in a short period of time by performance of T7 assays on injected fish and would not require additional reagents.
    2. The authors claim that there were no structural defects observed in urp1/2 double mutants. However, the hemal arch in figure 3 E seems to be deformed. This could be normal variance or a phenotype. This can be addressed by simple reinspection of the scans.

    Minor points:

    1. It would be helpful to the reader to add a little more information on urp1 and upr2 proteins and their processing to make it clear while only the 3' region of the protein was targeted to induce mutations.
    2. It would also be helpful to the reader to have a schematic indicating the guide target sites (they could be added to figure S1 C + D) in the protein to be able to interpret the result more easily.
    3. Figure 5: Addition of a square to H would help understand were the pictures in D-F were taken.

    Significance

    While scoliosis in human patients is very prevalent, our understanding on the mechanism that lead to the development of spinal curvature are very limited and so are the treatment strategies. The zebrafish has emerged as an important model to study spine development and formation of scoliosis. While not all findings in the presented work are novel, this work adds important information on the role of urotensin signaling in spine formation. These findings are of special significance in the light of recent reports that mutations in UTS2R, the human ortholog of uts2r3, show association with spinal curvature in patients with adolescent idiopathic scoliosis. As such, this work will be of interest not only to basic researches but also the medical field.

    My field of expertise: zebrafish, CRISPR/Cas, genetics, skeletal development, spine formation

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    Referee #2

    Evidence, reproducibility and clarity

    Major concern:

    1. Need to show that the CRISPANT targeting was effective for mutagenesis at each loci screened in the work presented in Figure 1E.

    Minor Concern:

    1. Fig 3F : might be improved by making the images black and white and possibly inverted. It is not easy to clearly see the vertebrae as is.

    Significance

    Summary:

    This is a beautifully executed study on the role of Urp signaling in spine morphogenesis in zebrafish. This work also challenges the model that Urp1/ 2 controls the extension and straightening of the body axis of the zebrafish embryos. Here, using a double mutant in urp1 and urp2, they show that urp1/2 are dispensable for axial straightening. Moreover, they provide redundant roles during larval development in particular for maintaining a straight spine. They go on to show that scoliosis observed in urp1/2 double mutant fish are distinct - showing only dorsal-ventral lordosis , whereas previously published scoliosis phenotypes _showing curvates in dorsal-ventral and medial-lateral axes as observed in cilia- and Reissner fiber-related scoliosis mutants. They provide clear evidence that loss of Urp signaling does not affect the stability of the Reissner fiber as it does in cilia-related scoliosis mutants. Underscoring the distinct regulation of Urp signaling on spine morphology during larval development. Altogether, this is an exciting advance in our understanding of one of the essential signaling pathways that help to regulate body axis straightening and spine morphogenesis in zebrafish. These studies may also help to further our understanding of the etiology and pathophysiology of multiple forms of neuromuscular scoliosis in humans. I recommend it for publication after revisions.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The authors investigate the role of Urotensin Related Peptides (Urp1 and Urp2) on zebrafish spine straightness. One model of normal spinal morphogenesis proposes that when the spine bends, material in the central canal of the spinal cord (the Reissner Fiber, RF, mostly composed of scospondin) stimulates surrounding Cerebral Spinal Fluid contacting neurons (CSF-cN), that in turn release Urotensin like peptides that cause dorsl muscles to contract and straighten the spine. It is clear that motile cilia in the central canal are responsible for forming/compacting the RF from monomers of scospondin. Mutations were generated that removed the peptide-coding portion of Urp1, Urp2 and that removed most of the Urp receptor Uts2r3 and made a missense scospondin gene. They used cfap298-/- as an immotile cilia control and scospondin-/- as a Reissner Fiber absent control. The authors show Urotensin peptides and receptor Uts2r3 function in juvenile but not embryonic axis straightening. They defined the timecourse of spinal curves onset and change during larval life, i.e., 9 dpf to 17 dpf and found that curves were dynamic between 30dpf and 3 mpf. Unlike cfap mutants, urotensin mutants show no sex bias in scoliosis expression. The authors used a temperature sensitive mutation in cfap298 and the GFP-tagged scospondin gene to show that active cilia are required for both initial formation of the RF before 28 hpf and to maintain the RF between 6 and 12 dpf. Finally the authors demonstrated that the receptor Uts2r3 is not required for establishment or maintenance of the RF at 28 hpf and 12 dpf.

    Major comments

    The key conclusions are well supported, see below for my two major issues.

    1. Please don't call this lordosis. Lordosis or hyperlordosis effects lumbar vertebra. The curve in the lumbar region shifts body weight so that human gait is more efficient that that in the great apes, or so the story goes. Zebrafish do not have lumbar vertebra equivalents or a natural curve in the caudal region. Similarly, fish do not have the equivalent vertebra to generate kyphosis, which is again a hyper flexion of a normal human spinal curve. Instead zebrafish have Weberian, precaudal and caudal vertebra. It would be so much more useful for the field if the authors used these terms and specified ranges, i.e. numbered vertebrae, that are effected so we can directly and accurately compare regions of defects between zebrafish mutants. It would help to make the point that the uts2r3 mutant has more caudally located curves than urp1/2 double mutants.
    2. The observation that urp1/2 double mutants have curves only in the D/V plane and almost completely lack side-to-side curves is noteworthy. Does the urp1-/-urp2-/- mutant uncouple two systems for posture? If this separate a DV from side-to-side postural control system, that would be very interesting. It is particularly important to describe how penetrant the phenotype is and how many times it was observed. See 9 minor comments. It would help the reader if the authors explicitly described the features that they see in the cfap298 mutant that constitute lateral curves and that are lacking in urp1/2 (e.g. in figure 4E).

    Minor comments

    1. Figure 1D legend says urp1 is expressed in dorsal while urp2 is express in all CSF-cNeurons, but the image for urp1 shows only ventral cells in WT, while the image for urp2 shows the same cells ...and more dorsal cells. Please replace image with one that matches the text.
    2. In Figure 2H, the position of curve apex graphic, how many fish were examined? In 2f it looks like n=8 and n=9. Can this info be added to the figure?
    3. Supplementary FigS3B How to measure the Cobb Angle is unclear. Why is the first curve not counted? I count 3 curves. First a ventral displacement, then a dorsal to ventral return, then a sharp flex before the tail. How to measure Cobb angle might be easier to explain if the figure is expanded into steps. Identify the apical vertebra, then showing how the lines are drawn parallel to those vertebrae, then where the measured angle forms between the lines perpendicular to the drawn parallel lines.
    4. I did not find legends for the movies. The first call to the movies calls movies 1-3 without explaining what each shows. The labels on the downloaded files are not informative.
    5. a. I think we (zebrafish biologists) need be explicit about what we mean with "without vertebral defects." What do we count as defects? Vertebrae can be fused, bent, shortened or the growing edges can be slanted. In Figure 3E, and movie7, it is clear that the highlighted mutant vertebrae are shorter than WT. The growing ends of normal vertebra are perpendicular to the long axis of the vertebra. In the mutants the ends are slanted. Please define in the text what you consider a relevant vertebral defect, because these vertebrae have defects. Or are you only considering the calcein stained centra at 10dpf?

    5b. Do you want to base your patterning conclusion on primarily the calcein data as these are closer to the notochord patterning time window. Please anchor this conclusion to a specific time or standard length e.g. 10dpf/5.6mm.

    1. "At 30 dpf... several mutants exhibited a significant curve in the pre-caudal vertebrae, in addition to a caudal curve (Fig. 3D and S3C). Since pre-caudal curves were rare in mutants at 3-months, this suggested that curve location is dynamic" The frequency of this observation is important. Does it effect all or a fraction of mutants? Can you provide some numbers to anchor these observations? Maybe fractions e.g.. 3 of 4 fish had precaudal curves at 30pdf, and 0 of 10 fish had precaudal curves by 3 mpf?
    2. The description of the pkd2l1 mutant, instead of terming it kyphosis can you tell the reader the vertebra number at the peak of the curve. The authors say the pkd2l1 mutant is highly distinct from urp1/urp2-/-, but the reader needs to hear exactly what is distinct. For example, does this mutant have both lateral and D/V curves?
    3. Does uts2r3-/- have no /minimal side-to-side curves like urp1/urp2-/-?
    4. One finding that deserves more discussion is the observation that urp1/urp2 double mutants have almost no side-to-side defects and all the obvious bends are in the D/V plane. Does this uncouple two systems for posture? Please consider the following paper. It shows a proprioception system that maintains normal side-to-side posture. A spinal organ of proprioception for integrated motor action feedback. Picton LD, Bertuzzi M, Pallucchi I, Fontanel P, Dahlberg E, Björnfors ER, Iacoviello F, Shearing PR, El Manira A. Neuron. 2021 Apr 7;109(7):1188-1201.e7. doi: 10.1016/j.neuron.2021.01.018. Epub 2021 Feb 11. PMID: 33577748

    Significance

    Scoliosis effects about 3% of children worldwide. Mammals have not been good models for this condition. Zebrafish seem to have an intrinsic susceptibility to scoliosis, as well as several technical advantages. Scoliosis is likely caused by disruption of several different and independent pathways. Building a model that describes one of these pathways will allow us to begin to test for therapies to treat or prevent scoliosis.

    1. The authors demonstrate that urp1 and 2 are required for normal adult spine straightness. While loss of the uts2r3 receptor (A.K.A. uts2ra, Zhang et.al., Nat Genet, 2018) and the uts4 (receptor, Alejevski, et.al, Open Bio, 2021) lead to adult spinal bends or scoliosis, of the four described urotensin ligand paralogs, only urp, not uts2, urp1 or urp2 have been tested by deletion for a role in scoliosis (Quan et.al., Peptides 2021). In the current work, the authors help to build a larger model of normal spine morphogenesis and show that mutations effecting later steps do not have typical cilia associated phenotypes. Contributing a step to this model is important.
    2. The authors show that juvenile or adult scoliosis can be independent of the embryonic curves, Curly Tail Down phenotype. This result is somewhat in conflict with previous work from Zhang, in which Curly Tail Down phenotype from a cilia defective mutant (ZMYND10) was rescued by overexpression of urp1 peptide. It is possible that urp1 functions in place of the natural peptide for this function. As before there are four paralogs of urotensin peptides. The second conflicting observation from Zhang is that embryos injected with morpholino to urp1 shows Curly Tail Down phenotype. It is well known that morpholinos can have off-target effects.
    3. The authors observe that urp1/urp2 double mutants have almost no side-to-side defects and all the obvious bends are in the D/V plane. Does this uncouple two systems for posture? If this separate a DV from side-to-side postural control system, that would be amazing.
    4. The authors provide evidence that curves are dynamic and erasable between 30 dpf and 3 mpf. This could be a time window to apply therapeutics.
    5. The authors provide a new graphic tool, a chart that logs the location of the apical curve vertebra (Figure 2H and SFigure 3C). This will allow better comparison between various scoliosis mutants.
    6. The authors describe 3 different version of scoliosis in 3 mutants. In cfap298 mutants (immotile cilia) curves effect all 3 dimensions. In urp1/urp2-/- mutants, curves only appear in the D/V plane. In uts2r3 mutants, curves appear more caudal than those in urp1-/-,urp2-/- mutants, though it is not clear if these are 3D curves.

    Audience: Biologists and physicians interested in 1) scoliosis, 2) normal morphogenesis, and 3) maintenance of the spine, 4)neurophysiologists interested in postural control and regulation of repetitive movements, like walking and swimming.

    My expertise: zebrafish genetics, scoliosis, gastrulation

  6. Version published to 10.1101/2022.08.13.503856 on bioRxiv