Unveiling Vertebrate Development Dynamics in Frog Xenopus laevis using Micro-CT Imaging

  1. Laznovsky Jakub
  2. Kavkova Michaela
  3. Reis Alice
  4. Robovska-Havelkova Pavla
  5. Krivanek Jan
  6. Zikmund Tomas
  7. Kaiser Jozef
  8. Buchtova Marcela
  9. Harnos Jakub

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Abstract

Background

Xenopus laevis , the African clawed frog, is a versatile vertebrate model organism employed across various biological disciplines, prominently in developmental biology to elucidate the intricate processes underpinning body plan reorganization during metamorphosis. Despite its widespread utility, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus’ late developmental stages.

Findings

In the present study, we harnessed micro-computed tomography (micro-CT), a non-invasive 3D imaging technique utilizing X-rays to examine structures at a micrometer scale, to investigate the developmental dynamics and morphological changes of this crucial vertebrate model. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adult frogs. This extensive dataset enhances our understanding of vertebrate development and is adaptable for various analyses. For instance, we conducted a thorough examination, analyzing body size, shape, and morphological features, with a specific emphasis on skeletogenesis, teeth, and organs like the brain at different stages. Our analysis yielded valuable insights into the morphological changes and structure dynamics in 3D space during Xenopus’ development, some of which were not previously documented in such meticulous detail. This implies that our datasets effectively capture and thoroughly examine Xenopus specimens. Thus, these datasets hold the solid potential for additional morphological and morphometric analyses, including individual segmentation of both hard and soft tissue elements within Xenopus .

Conclusions

Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus’ development and the associated morphological changes. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans have the potential for use in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.

Graphical abstract & lay summary

3D images of selected developmental stages of X. laevis in a comparison (scale bar = 10 mm).

Lay summary

X-ray tomography was used to examine the African clawed frog ( Xenopus laevis ). An extensive data set of specimens from tadpoles to adult frogs provides novel insights into the changes and developmental dynamics of selected structures, which opens avenues to an improved understanding of this crucial animal model.

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  1. GigaScience

    Background Xenopus laevis, the African clawed frog, is a versatile vertebrate model organism employed across various biological disciplines, prominently in developmental biology to elucidate the intricate processes underpinning body plan reorganization during metamorphosis. Despite its widespread utility, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus’ late developmental stages.Findings In the present study, we harnessed micro-computed tomography (micro-CT), a non-invasive 3D imaging technique utilizing X-rays to examine structures at a micrometer scale, to investigate the developmental dynamics and morphological changes of this crucial vertebrate model. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adult frogs. This extensive dataset enhances our understanding of vertebrate development and is adaptable for various analyses. For instance, we conducted a thorough examination, analyzing body size, shape, and morphological features, with a specific emphasis on skeletogenesis, teeth, and organs like the brain at different stages. Our analysis yielded valuable insights into the morphological changes and structure dynamics in 3D space during Xenopus’ development, some of which were not previously documented in such meticulous detail. This implies that our datasets effectively capture and thoroughly examine Xenopus specimens. Thus, these datasets hold the solid potential for additional morphological and morphometric analyses, including individual segmentation of both hard and soft tissue elements within Xenopus.Conclusions Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus’ development and the associated morphological changes. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans have the potential for use in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.

    This work has been peer reviewed in GigaScience (see paper), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

    Reviewer name: Virgilio Gail Ponferrada (R1)

    Thanks to the authors for accommodating the reviewers' suggestions. The manuscript continues to be well constructed and easy to read. I appreciate the addition of micro-CT analysis of Xenopus gut development and the inclusion of scans of additional samples for statistical analysis bolstering their findings. Should the manuscript be accepted for publication, perhaps the authors will contact Xenbase (www.xenbase.org), the Xenopus research database, as an additional means of featuring their micro-CT datasets. I suggest this manuscript be accepted for publication.

  2. GigaScience

    Background Xenopus laevis, the African clawed frog, is a versatile vertebrate model organism employed across various biological disciplines, prominently in developmental biology to elucidate the intricate processes underpinning body plan reorganization during metamorphosis. Despite its widespread utility, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus’ late developmental stages.Findings In the present study, we harnessed micro-computed tomography (micro-CT), a non-invasive 3D imaging technique utilizing X-rays to examine structures at a micrometer scale, to investigate the developmental dynamics and morphological changes of this crucial vertebrate model. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adult frogs. This extensive dataset enhances our understanding of vertebrate development and is adaptable for various analyses. For instance, we conducted a thorough examination, analyzing body size, shape, and morphological features, with a specific emphasis on skeletogenesis, teeth, and organs like the brain at different stages. Our analysis yielded valuable insights into the morphological changes and structure dynamics in 3D space during Xenopus’ development, some of which were not previously documented in such meticulous detail. This implies that our datasets effectively capture and thoroughly examine Xenopus specimens. Thus, these datasets hold the solid potential for additional morphological and morphometric analyses, including individual segmentation of both hard and soft tissue elements within Xenopus.Conclusions Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus’ development and the associated morphological changes. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans have the potential for use in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.

    This work has been peer reviewed in GigaScience (see paper), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

    Reviewer name: **John Wallingford **(Original submission)

    Laznovsky et al. present a nice compendium of micro-CT-based digital volumes of several stages of Xenopus development. Given the prominence of this important model animal in studies of developmental biology and physiology, this dataset is quite useful and will be of interest to the community. That said, the study has some key limitations that will limit its utility for the research community, though these do not reduce the dataset's impact in the education and popular science realms, which is also a stated goal for the paper. Overall, I recommend publication after an effort has been made to address the following concerns.

    1. The atlas adequately samples developmental stages from late tadpole through metamorphosis. However, as far as I can tell only a single sample has been imaged at each stage. Thus, the quantifications of inter-stage differences shown here (Fig. 2, 4, 5) are at best very rough estimates and also provide no information about intra-stage variability in these metrics. This is not a fatal weakness, but it is an important caveat that I believe should be very explicitly stated in the text and in the figure legend of relevant figures.

    2. I am very disappointed that the rich history of microCT on Xenopus seems to have been entirely ignored by these authors. MicroCT has already been used to describe the skull, the brain, liver, blood vessels, etc. during Xenopus development. (Just a few papers the authors should read are: Slater et al., PLoS One 2009; Senevirathnea et al., PNAS, 2019; Ishii et al., Dev. Growth, Diff. 2023; Zhu et al., Front. Zool 2020.) It has also been used for comparative studies of other frogs (Kondo et al., Dev. Growth, Diff. 2022; Kraus, Anat. Rec. 2021; Jandausch et al., Zool. Anz. 2022; Paluh, et al., Evolution 2021, Paluh et al., eLife 2021). None of these -or the many other relevant papers- are discussed or cited here. The research community would be much better served if authors make a serious effort to integrate their methods and their results into this existing literature.

    3. An opportunity may have been missed here to provide some truly new biological insights: The gut remodels substantially during metamorphosis, but to my knowledge that has NOT be previously examined by microCT. It may not work, as the gut may simply be too soft to visualize, but then again, it may be worth trying.

  3. GigaScience

    Background Xenopus laevis, the African clawed frog, is a versatile vertebrate model organism employed across various biological disciplines, prominently in developmental biology to elucidate the intricate processes underpinning body plan reorganization during metamorphosis. Despite its widespread utility, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus’ late developmental stages.Findings In the present study, we harnessed micro-computed tomography (micro-CT), a non-invasive 3D imaging technique utilizing X-rays to examine structures at a micrometer scale, to investigate the developmental dynamics and morphological changes of this crucial vertebrate model. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adult frogs. This extensive dataset enhances our understanding of vertebrate development and is adaptable for various analyses. For instance, we conducted a thorough examination, analyzing body size, shape, and morphological features, with a specific emphasis on skeletogenesis, teeth, and organs like the brain at different stages. Our analysis yielded valuable insights into the morphological changes and structure dynamics in 3D space during Xenopus’ development, some of which were not previously documented in such meticulous detail. This implies that our datasets effectively capture and thoroughly examine Xenopus specimens. Thus, these datasets hold the solid potential for additional morphological and morphometric analyses, including individual segmentation of both hard and soft tissue elements within Xenopus.Conclusions Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus’ development and the associated morphological changes. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans have the potential for use in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.

    This work has been peer reviewed in GigaScience (see paper), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

    Reviewer name: Virgilio Gail Ponferrada (Original submission)

    The manuscript is well written and easy to understand. It will be a good contribution to the Xenopus research community as well as a useful reference for the field of developmental and amphibian biology.

    I suggest the following revisions:

    • For the graphical abstract try alternating NF stage numbers above and below samples for a cleaner look, adult male and adult female can both remain at the top.
    • Appreciate the rationale for providing the microCT analysis presented in this manuscript and choices of late stage tadpoles, pre- and prometamorphosis, through metamporphosis to the adult male and female frog.
    • For the head development section authors can make reference to the Xenhead drawings, Zahn et al. Development 2017.
    • Head Development section paragraph 4, change word from "gender" to "sex."
    • Supplementary Table 3. Change "gender-related" to "sex-related."
    • Micro-CT Data Analysis of Long Bone Growth Dynamics section paragraph 1 change "in terms of gender" to "in terms of sex."
    • Figure 4 panels A and B don't reflect the observation that adult females are enlarged males. While the authors state that the view of the male and female skeletons are maximized and not proportional as stated in the caption, suggest that scale bars be employed and the images adjusted to show the size relationship difference between the sexes as in Figure 1. On first glance and perhaps to those not as familiar with the difference in sex size in Xenopus that in this particular example of the adult male image being more spread out compared to the image of the female, it feels misleading. - Ossification Analysis section paragraph 2 change "frog's gender" to "frog's sex."
    • Figure 5 panel A, the label is overlapping "NF 59." For panels B and B' scale bars on these panels would help the reader understand the proportions. Yes, there is the 3mm scale bar from panel A and as stated in the caption, but including them in the B panels could help even if panel B had a scale bar labeled at 0.25 mm and panel B' was 3 mm.
    • Segmentation of Selected Internal Soft Organ section, perhaps more commentary on the ability to observe the development of the segmentation of the brain regions: cbh: cerebral hemispheres; cbl: cerebellum; dch: diencephalon; mob: medulla oblongata; opl: optic lobes; sp: spinal cord while clearly shown in Figure 6, some accompanying description in the text would help readers in general or give the implication that microCT analysis of mutant or diseased frogs could help identify physical characteristics of frogs with developmental or neurological disorders. This would help transition from the analysis of a specific organ to the next section Further Biological Potential of Xenopus's Data.
    • These analyses, while thorough accompanied by novel visuals, require statistical implementation of multiple tadpoles and frogs per NF stage to account for variation in samples and to bolster the claims stated in skull thickness, the head mass and eye distance changes, increased length of the long bones during maturation, and femural ossification cartilage to bone ratios. This may constitute a suggested major revision to perform these analyses.
  4. GigaScience

    Background Xenopus laevis, the African clawed frog, is a versatile vertebrate model organism employed across various biological disciplines, prominently in developmental biology to elucidate the intricate processes underpinning body plan reorganization during metamorphosis. Despite its widespread utility, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus’ late developmental stages.Findings In the present study, we harnessed micro-computed tomography (micro-CT), a non-invasive 3D imaging technique utilizing X-rays to examine structures at a micrometer scale, to investigate the developmental dynamics and morphological changes of this crucial vertebrate model. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adult frogs. This extensive dataset enhances our understanding of vertebrate development and is adaptable for various analyses. For instance, we conducted a thorough examination, analyzing body size, shape, and morphological features, with a specific emphasis on skeletogenesis, teeth, and organs like the brain at different stages. Our analysis yielded valuable insights into the morphological changes and structure dynamics in 3D space during Xenopus’ development, some of which were not previously documented in such meticulous detail. This implies that our datasets effectively capture and thoroughly examine Xenopus specimens. Thus, these datasets hold the solid potential for additional morphological and morphometric analyses, including individual segmentation of both hard and soft tissue elements within Xenopus.Conclusions Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus’ development and the associated morphological changes. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans have the potential for use in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.

    This work has been peer reviewed in GigaScience (see paper), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

    Reviewer name: **Brian Metscher **(Original submission)

    The authors present a set of 3D images of selected developmental stages of the widely-used laboratory model Xenopus laevis along with some examples of how the data might be used in developmental analyses. The dataset covers stages from mid-larva through metamorphosis to adult, which should provide a starting point for various studies of morphological development. Some studies will undoubtedly require other stages or more detailed images, but the presented data were collected with straightforward methods that will allow compatibility with future work.

    The data appear to be sound in the collection and curation. Data availability is made clear in the article, and the complete set will be publicly available in standard formats on the Zenodo repository. This should ensure full accessibility to anyone interested. The article is well-organized and clearly written.

    A few points about the methods could be clarified: Was only one specimen per stage scanned? Specimens were dehydrated through an ethanol series and then stained with free iodine in 90% methanol, and then rehydrated back through ethanol. Why was methanol used for the staining and not dehydration? It seems odd to switch alcohols back and forth without intermediate steps. This could have some effect on tissue shrinkage. It should be indicated that the X-ray source target is tungsten (even though it is unlikely to be anything else in this machine). The "real images" (p. 7) in Suppl. Fig. 1 should simply be called photographs - microCT images are real too. For the measurements of bone mass, is the cartilage itself actually visible in the microCT images? p. 13: "The dataset's diverse species representation…" What does this mean? It is only one species. The limitations on the image data are not discussed. All images have limits to their useful resolution and contrast among components; this is not a weakness, just a reality of imaging. The different reconstructed voxel sizes for different size specimens are mentioned, but it might be helpful to indicate the voxel sizes in Figure 1 as well as in the relevant table. And if the middle column of Figure 1 could be published with full resolution of the snapshots it would help show the actual quality of the images.

  5. Version published to 10.1101/2024.06.11.598452v1 on bioRxiv