Unveiling vertebrate development dynamics in frog Xenopus laevis using micro-CT imaging

GigaScience , 2024, 13 , 1–16 DOI: 10.1093/gigascience/giae037 Research Unv eiling v ertebrate dev elopment dynamics in frog Xenopus laevis using micro-CT imaging J akub Laznovsk y 1 , Michaela Kavkov a 1 , Alice Helena Reis 2 , P avla Robo vska-Havelko va 3 , Lorena Agostini Maia 4 , J an Kri v anek 5 , Tomas Zikm und 1 , J ozef Kaiser 1 ,6 , Mar cela Buc htov a 4 ,7 , and J akub Harnos 4 , * 1 Centr al Eur opean Institute of Tec hnology, Brno Univ ersity of Tec hnology, 612 00 Brno, Czech Republic 2 Department of Chemical Engineering, Columbia Univ ersity, Ne w York, NY 10025, USA, and Department of Genetics and De v elopment, Columbia Stem Cell Initiati ve, Columbia Uni versity Irving Medical Center, New York, NY 10032, USA 3 Department of Zoology, Faculty of Science, University of South Bohemia, 370 05 Ceske Budejovice, Czech Republic 4 Department of Experimental Biology, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic 5 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 625 00 Brno, Czech Republic 6 Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, 616 69 Brno, Czech Republic 7 Laboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, v .v .i., Czech Academy of Sciences, 602 00 Brno, Czech Republic ∗Corr espondence addr ess . Jakub Harnos , Masaryk University Faculty of Science Bohunice Campus Building D36, Kamenice 5, Brno 62500, Czech Republic. E-mail: [email protected] Abstract Bac kgr ound: Xenopus laevis , the African clawed frog, is a v ersatile v ertebrate model organism in various biological disciplines, prominently in developmental biology to study body plan reorganization during metamorphosis. However, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus ’ late developmental stages. Findings: This study utilized micr o–computed tomography (micr o-CT), a noninv asi v e 3-dimensional (3D) ima ging technique with micr ometer-scale r esolution, to explor e the dev elopmental dynamics and morpholog ical chang es in Xenopus laevis . Our approach inv olv ed generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adults. This dataset enhances our understanding of vertebrate development and supports various analyses. We conducted a careful examination, analyzing body size , shape , and morphological features, focusing on skeletogenesis, teeth, and organs like the brain and gut at different stages. Our analysis yielded valuable insights into 3D morphological changes during Xenopus ’ development, documenting details pr eviousl y unr ecorded. These datasets hold the solid potential for further morphological and morphometric analyses, including segmentation of hard and soft tissues. Conclusions: Our r e positor y of micr o-CT scans r e pr esents a significant r esource that can enhance our understanding of Xenopus ’ development and the associated morphological changes in the future. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a compr ehensi v e series of dev elopmental sta ges, opens up extensi v e opportunities for their br oader r esear c h application. Mor eov er, these scans can be used in virtual reality, 3D printing, and educational conte xts, further e xpanding their value and impact. Gr aphical abstr act 3D computed images of selected developmental stages of X. laevis. Summary : X-r ay tomogr aphy was used to examine the African clawed frog ( Xenopus laevis ). This extensi v e dataset of specimens from tadpoles to adult frogs opens av en ues to nov el insights into the changes and developmental dynamics of selected structures, leading ev entuall y to an impr ov ed understanding of this crucial animal model. Ke yw ords: Xenopus laevis , development, vertebrates, micro–computed tomography, morpholog ical chang es Recei v ed: November 30, 2023. Revised: April 10, 2024. Accepted: June 3, 2024 ©The Author(s) 2024. Published by Oxford Uni v ersity Pr ess on behalf of GigaScience. This is an Open Access article distributed under the terms of the Cr eati v e Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024 2 | GigaScience , 2024, Vol. 13 Bac kgr ound Xenopus laevis (NCBI:txid8355), commonly known as the African clawed fr og, serv es as a fundamental model or ganism in the field of life sciences. Its widespr ead adoption, particularl y in biological r esearc h, can be attributed to the ease of br eeding and housing, as well as the substantial size and manipulability of its eggs and embryos [ 1 , 2 ]. Over the years, this species has been extensiv el y inv estigated acr oss v arious disciplines , including genetics , embryology, and de v elopmental, cell, and r egener ativ e biology [ 3– 5 ]. While initial attempts at anatomical descriptions of X. laevis date back a century [ 6 ], subsequent studies ha ve , to varying degr ees, concentr ated onl y on specific anatomical regions [ 7– 25 ]. Despite these valuable contributions, many past approaches to describing frog anatomy failed to preserve the intricate morphological details, making them unsuitable for detailed and compar ativ e studies. In response to these limitations, a detailed description of an adult male frog’s anatomy as a complete organism using a nondestructive micro–computed tomography (microCT) method was r ecentl y pr ovided [ 7 ]. Micr o-CT, to be candid, has already been emplo y ed to some extent as a valuable tool in selected stages of X. laevis ’ embryos , tadpoles , and frogs to explore localized or specific occurrences, such as gastrulation [ 26 ], craniofacial [ 27 ] and urostyle [ 28 ] development, skeletal morphology [ 29 , 30 ], limb (skeletal) r egener ation [ 31–33 ], br ain r egener ation [ 34 ], and the anatomy of cranial and anterior spinal nerves [ 35 ], the head [ 36 , 37 ], and se v er al internal organs [ 38 ]. Besides X. laevis itself, micro-CT has also been utilized in closel y r elated frogs to in vestigate , for instance , c hondr ocr anium or ganization in Alytes obstetricans [ 39 ], skeleton [ 40 ] and digit [ 41 ] morphology in Xenopus tropicalis , cortical bone morphology in various anuran amphibians [ 42 ], and reevolution of lost mandibular teeth in Gastrotheca guentheri [ 43 , 44 ], together with cr anial m usculoskeletal structures in Pelobates fuscus [ 45 ], lung rearrangements during metamorphosis in Microhyla fissipes [ 46 ], gill formation during metamorphosis in Bufo bufo [ 47 ], and metabolic r eor ganization in metamorphic Rana omeimontis tadpoles [ 48 ]. Nevertheless, the spatiotemporal dynamics of X. laevis ’ late development at the le v el of a whole organism, along with the comparison between adult male and female fr ogs, r emain lar gel y unexplor ed in sufficient detail. To fill this knowledge gap and gain a more comprehensive understanding of X. laevis ’ late de v elopment at the le v el of a whole organism, we employed micro-CT, a powerful imaging technology for examining small objects at the micrometer scale [ 49 , 50 ]. Micr o-CT utilizes X-r ays to generate high-resolution 3dimensional (3D) images, making it an ideal tool for investigating de v elopmental and mor phological c hanges, particularl y in embryos , tadpoles , and small animals [ 49 , 50 ]. In principle, micro-CT imaging is based on capturing a series of 2-dimensional (2D) X-ray r adiogr a phs fr om v arious angles and then mathematicall y pr ocessing them through tomographic reconstruction, resulting in a 3D matrix r epr esenting volume density. An adv anta ge of micr o-CT lies in its ability to image bones and, when combined with various contrast methods, also soft tissues and blood vessels within the same sample [ 49 , 50 ]. Thr ough micr o-CT, we pr esent high-r esolution ima ges and computed 3D models of de v eloping X. laevis tadpoles , froglets , and adult fr ogs, whic h offer gr eat potential for r esearc h fields like de v elopmental and compar ativ e biology. Additionall y, we conducted preliminary analyses of this dataset to illustrate its versatility . Specifically , our preliminary examination covered morphological changes in body size , shape , and skeletogenesis from selected pr emetamor phosis, pr ometamor phosis, and climax metamor phosis sta ges thr ough the fr oglet sta ge up to adulthood. Our findings unveil promising and opening insights into the intricate morphological dynamics of late X. laevis ’ development. To sum up, this r esearc h contributes a unique dataset concerning the de v elopmental and mor phological c hanges in X. laevis , including the adult stages, shedding light on the dynamics of v ertebr ate development and bearing broader implications for future analyses of Xenopus . Data Description/Sampling Strategy In our micro-CT study of Xenopus , we employed a careful sampling strategy to encompass the entire developmental spectrum of this amphibian species . T he selection of de v elopmental sta ges was based on specific mor phological featur es and markers that serve as critical indicators of X. laevis ’ development (see below). By str ategicall y c hoosing 9 distinct de v elopmental sta ges, we aimed to ca ptur e the compr ehensiv e pr ocess of mor phogenesis, spanning fr om pr emetamor phosis tadpoles to full y matur e adult fr ogs. These stages were carefully chosen with reference to specific criteria, such as the presence and dimensions of legs and the tail, or the r earr angements of the head, as outlined in the tables of Nieuwkoop and Faber [ 51 ] and Zahn and colleagues [ 52 ]. Our sampling included the following crucial stages of X. laevis ’ de v elopment: Premetamorphosis (stages 44–45, 52, and 53, according to Nieuwkoop and Faber, NF): These early stages provide insight into the initial stages of development on metamorphosis, as Xenopus tr ansforms fr om tadpole to fr oglets. Pr ometamorphosis (NF sta ges 54 and 57): This phase r epr esents an intermediate stage where significant changes in the head, limb buds, and tail are occurring, leading slowly to w ar d the climax metamorphosis. Climax metamorphosis (NF stages 59 and 62): These stages are c har acterized by the most profound changes, marking the peak of metamorphosis. Fr oglets (NF sta ge 66): Fr oglets r epr esent a tr ansitional sta ge, bridging the gap between tadpoles and fully mature adult frogs. Adult male and female frogs : These adult stages represent the endpoint of Xenopus de v elopment, allowing us to observe fully mature individuals and to compare differences between sexes. We c hose or ganisms that wer e a ppr oximatel y 1 year old for this micro-CT study. Our selection of these k e y de v elopmental sta ges was performed with regard to their significance in the overall developmental process of X. laevis . By examining these specific stages, we aimed to gain a solid dataset, enabling a compr ehensiv e understanding of the mor phological tr ansformations and de v elopmental milestones that occur during X. laevis ’ de v elopment, fr om its primary aquatic tadpole phase to its secondary aquatic adult frog stage [ 53 ]. This sampling strategy provided a detailed and holistic view of X. laevis ’ late de v elopment, enabling us to perform valuable analyses leading to preliminary insights and conclusions about this species’ morphological changes throughout its life cycle. Methods/Source of Samples Xenopus embry os w er e gener ated and cultiv ated following standard protocols. Briefly, testes wer e sur gicall y r emov ed fr om anesthetized males (20% MS-222, Sigma-Aldrich, A5040) and transferred to cold 1 ×Marc’s Modified Ringers (MMR; 100 mM NaCl, Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024 Unv eiling v ertebr ate de v elopment dynamics in fr og | 3 2 mM KCl, 1 mM MgSO 4 , 2 mM CaCl 2 , 5 mM HEPES, buffered to pH 7.4), supplemented with 50 μg/mL gentamycin (Sigma-Aldrich, G3632). To induce egg laying, fully mature Xenopus females were injected with 260 U of human c horionic gonadotr opin (Merc k, Ovitrelle 250 G) into the dorsal lymph sac a ppr oximatel y 12 to 16 hours before use and were k e pt overnight at 18 ◦C. For fertilization, eggs were extracted from induced females directly into a Petri dish and mixed with a piece of testes in 0.1 ×MMR at 18–21 ◦C. The subsequent de v elopment of Xenopus tadpoles, fr oglets, and adult frogs adhered to the approved cultivation protocols (see abo ve). T he Xenopus froglets and adult frogs were reared in XenopLus (catalog number RE18001301, Tecniplast), whic h is an adv anced, full y automated system tailored for housing amphibians, guaranteeing excellent animal care and housing conditions. At a designated time point, Xenopus specimens were anesthetized and fixed in a buffer ed 4% par aformaldehyde solution (1004965000, Merc k) for 3 hours (tadpoles) or overnight (froglets and adult frogs) and staged according to the tables of Nieuwkoop and Faber [ 51 ] and Zahn and collea gues [ 52 ]. Photogr a phs of selected Xenopus specimens used for micro-CT analysis are presented in Supplementary Fig. S1 . Methods/Micro-CT Scanning Prior to scanning, samples were placed in either a 2-mL Eppendorf tube, a 15-mL/50-mL Falcon tube (depending on sample size), or, in the case of the adult frogs, a 500-mL plastic container. To pr e v ent the motion and drying of the sample during the microCT scan, all samples were mounted in a 1% agarose gel (TopBIO, P045). Micr o-CT measur ements wer e conducted using the GE Phoenix v | tome | x L 240 laboratory system, which is equipped with a 180 kV/15 W nanofocus X-ray tube and a 4,000-pixel ×4,000pixel flat panel detector with a 100μm pixel size. Scan conditions for eac h sta ge ar e summarized in Supplementary Table S1 . It is important to note that the X-ray source target utilized in this study was tungsten. The subsequent tomogr a phic r econstruction was performed using the GE Phoenix datos | x 2.0 software . T he r econstructed data wer e imported into VG Studio MAX 2023.4 softwar e (Volume Gr a phics GmbH), in whic h the measur ed data wer e segmented, anal yzed, and visualized using VG Studio MAX ( RRID:SCR _ 017997 ). The segmentation of Xenopus guts was done manually by an operator using the software Avizo 2020.2 (Thermo Fisher ScientificA) (Avizo 3D Software, RRID:SCR _ 014431 ). All Xenopus samples wer e initiall y scanned in their native state to visualize bone structur es. Subsequentl y, specimens wer e stained with 1% iodine (Penta, 21210–11000) in a 90% methanol (Penta, 17570–30250) solution to enhance the contrast and visualize soft tissues. As for teeth (Fig. 3 ), the separated jaws were scanned and stained. The dehydration and staining times for eac h Xenopus de v elopmental sta ge ar e detailed in Supplementary Table S2 . Data Quality Control and Limitations All analyzed samples, comprising X. laevis tadpoles , froglets , and adult fr ogs, wer e uniforml y pr eserv ed and handled befor e scanning. The primary variable affecting data quality was the varied voxel size of each dataset, arising from different sample sizes. The smallest sample, a tadpole stage (NF 44–45), featured a voxel size of 5 μm, while the largest sample, an adult female frog, was scanned with a voxel size of 40 μm (for a voxel size of all scanned NF stages, see the appropriate column in Supplementary Table S1 or Fig. 1 ). The voxel size difference was dir ectl y corr elated with sample dimensions . T he GE L240 X-ray source’s cone beam geometry and the detector’s field of view allo w ed smaller samples to be placed closer to the X-ray source, resulting in a smaller voxel size (higher resolution) due to cone beam magnification. Although samples exhibited variable voxel sizes, this variability does not necessarily limit data analysis. Larger samples, despite having larger voxel sizes, still enabled the recognition of structures analyzed, as these structur es wer e pr oportionatel y lar ger in lar ger animals scanned with larger voxel sizes. All Xenopus specimens underwent 2 sequential scans. Initially, native scans were performed, following the principle of microCT imaging of samples in their native form without staining, enabling the visualization of dense miner alized structur es like bones and teeth. Subsequently, all scans were stained as described in Supplementary Table S2 . To pr e v ent mov ement and drying of samples during data acquisition, all samples were fixed in pol ypr opylene conical tubes with 1% a gar ose gel. Thr oughout data acquisition, no observable sample shrinkage occurred. If any negligible shrinkage or drying did occur, we anticipate the volume reduction to be pr oportionall y consistent across all Xenopus samples. Image data inherently exhibit limitations in terms of useful resolution and contrast among components . T hese constraints are integral to the imaging process and are not indicative of weaknesses in our study but rather intrinsic c har acteristics of the imaging modality emplo y ed. It is essential to acknowledge that, in any imaging technique, there are practical boundaries to the le v el of detail and contrast achievable. Atlas of X. laevis ’ Late De v elopment In order to provide a more comprehensive understanding of X. laevis ’ de v elopment during metamor phosis and or ganogenesis, we first collected specimens r epr esenting the key fr og sta ges, including pr emetamor phosis, pr ometamor phosis, climax metamorphosis , froglets , and adult male and female frogs (see Sampling Str ategy). Initiall y, we performed native sample scanning for hard tissue visualization and subsequently stained and scanned the samples for soft tissue visualization (see Micro-CT Scanning). As a result, we were able to collect and analyze all major stages of X. laevis ’ late de v elopment. The entir e collection of the Xenopus atlas is presented in Fig. 1 , and all raw data can be found in the GigaDB repository (see the Data Availability section) [ 54 ]. Subsequently, with this atlas , we pro vide here several examples of specific analyses of X. laevis ’ late de v elopment, including an analysis of head development, teeth, long bone growth dynamics, ossification, br ain de v elopment with its associated nerv es, and gut morphology. Head De v elopment Analysis First, we delved into the intriguing process of head development and skull metamorphosis in climax metamorphosis tadpoles, froglets, and adult frogs (refer to Fig. 2 A; Supplementary Video S1 ). Our initial investigation emplo y ed morphometric analysis to shed light on the de v elopmental c hanges in the skull. All skulls were oriented in the same direction (the anterior skull part heading to the top of a page; A: anterior; P: posterior; R: right; and L: left) in a top-bottom vie w. Suc h ima ges can be further used, for example, to e v aluate how the individual calvarian bones are being rearranged during metamorphosis or, for example, to shed light on facial region de v elopment. Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024 4 | GigaScience , 2024, Vol. 13 Figur e 1: T he atlas of Xenopus laevis ’ late de v elopment. (A) 3D r enders of soft tissues of selected Xenopus de v elopmental NF sta ges. (A  ) Cr oss sections of soft tissues . T he section le v el was selected based on capturing the brain area of Xenopus specimens. (A   ) 3D renders of the skeleton. In the first 3 conditions (NF 44–53), the bones are zoomed in and shown in the red rectangle on the left. The relevant scale bar and voxel size are depicted on bottom or top of each picture. All views are from dorsal view with the cranial side pointing to the right. A: anterior; L: left; P: posterior; R: right. Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024 Unv eiling v ertebr ate de v elopment dynamics in fr og | 5 Figur e 2: T he anal yses of the head de v elopment. (A) The series of Xenopus skulls of selected sta ges . T he scale bar with values in mm is shown on the left bottom. (B) The skull thickness of selected stages is displa yed. T he scale bar with values in mm is shown on the left side. (B  ) The r elativ e distribution of bone thickness for each skull is shown. The experiment involved conducting 3 repetitions for each developmental stage, with the results depicted as means accompanied by standard deviations ( ±SD). (C) The ov ervie w of de v eloping heads with highlighted eyes is shown. Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024 6 | GigaScience , 2024, Vol. 13 Figure 3: Demonstr ativ e inter activ e Sketc hFab visualizations of 3D r econstructions highlighting that Xenopus female heads exhibit an incr ease in volume compared to males. (A) Skull of Xenopus laevis adult female (link: https://sketchfab.com/3d-models/skull-of-xenopus-laevis-female321240df5a5741d39937f65d457c1594 ). (B) Skull of Xenopus laevis adult male (link: https://sketc hfab.com/3d-models/skull-of-xenopus-lae vis-male192dbbceb71f4b73a1097a7cdf67e5ae ). Next, we performed the examination of skull thickness (Fig. 2 B– B  ; Supplementary Video S2 ). This analysis revealed a nonlinear relationship, with adult frogs possessing significantly thicker skulls compared to their younger counterparts . T his in vestigation also suggests a positive correlation between skull mass and skull thickness. Moving on, we made an introductory analysis regarding the eye distance during de v elopment, as illustr ated in Fig. 2 C. Our mor phometric anal ysis (for an example of the measur ement, see Supplementary Fig. S2A ) r e v ealed a pr ogr essiv e decr ease in eye distance during de v elopmental sta ges (r oughl y estimated in Supplementary Fig. S2B –B  ). These c hanges actuall y occurr ed in Xenopus in 2 phases: a gradual increase in the premetamor phic sta ges (data not sho wn), follo w ed b y a peak at the onset of climax metamorphosis (NF stages 59 and 62), and finall y, a decr ease to a compact head configuration in froglets (NF stage 66) and adult frogs (Fig. 2 C and very roughly estimated in Supplementary Fig. S2B –B  ). This adaptation aligns well with the frog’s life strategy, transitioning from a w ater-dw elling tadpole with lateral eyes to an adult with eyes positioned on top of the head for a submerged lifestyle [ 55 ], reminiscent of crocodilians [ 56 ]. Based on the pr e vious par a gr a phs, one can also explor e the conserv ation anal ysis of the eye/head r atio with r espect to fr og sex. Findings of ours (and others [ 57 ]) indicate that Xenopus female heads exhibit an increase in volume compared to males (Fig. 3 A, B). Ho w e v er, although our effort was in no way directi ve, the e ye distance in females does not remain at 100% of the head volume in males, but it is r elativ el y less (for a preliminary estimate, see Supplementary Table S3 ). This observation, which can be even assessed also from the macroscopic scale , ma y be linked to visual perception r equir ements, as the proximity of the eyes is still crucial for certain aspects of vision [ 58 ]. Howe v er, our intention here was just to demonstrate that similar and pr ecise anal yses ar e feasible with micr o-CT data. In summary, our micro-CT dataset can be further emplo y ed for the study of the head and its associated organs such as the eyes in X. laevis . For more information about the X. laevis ’ head morphology in general, we also refer to the handmade drawings by Zahn and colleagues [ 52 ]. Tooth Analysis To enhance the depth of our analysis and increase its practical applicability, we conducted a comprehensive examination of the frog teeth, with an individual dataset of an adult female jaw. Upon initial observation, the presence of teeth in X. laevis may not be r eadil y a ppar ent. This is due to the fact that teeth were exclusiv el y found in the maxilla, r eferr ed to as maxillary teeth (Fig. 4 A, B) and behind the maxillary arch on the vomeral bone— so-called vomeral teeth (not shown). Additionall y, onl y a small portion of the maxillary tooth protruded into the oral cavity [ 59 ] (Fig. 4 C). In contrast, the mandible contains no teeth (Fig. 4 D). The shape of maxillary teeth was generally uniform (i.e., homodont dentition), typically resembling simple conical structures (see Fig. 4 B). Xenopus ’ teeth exhibit an acrodont type of attachment, where they form an ankylotic attachment with the adjacent bone and are characteristic by regular renewal, a condition kno wn as polyphy odont dentition. Notably, the phenomenon of tooth r ene wal in Xenopus is akin to what has been pr e viousl y documented in other species, such as geckos [ 60 , 61 ]. In agreement with this, we also noticed the evidence of the replacement in the maxillary teeth (Fig. 4 E). These anal yses r e v ealed the pr ogression of ankylosis, from the early development of teeth located closer to the g ing iva to their full fusion with the adjacent bone (see Fig. 4 E). Our Xenopus micro-CT dataset thus unveils the pr e viousl y concealed dental structur es of X. laevis , pr ovides a high-r esolution r e v elation of their “hidden” teeth patterning, and offers new perspectives for studying teeth and their growth in Xenopus . Micr o-CT Da ta Analysis of Long Bone Gr o wth Dynamics Next, we assessed the growth dynamics of se v er al long bones (shown in adult frogs in Fig. 5 A, B and Supplementary Video S3 ; Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024 Unv eiling v ertebr ate de v elopment dynamics in fr og | 7 Figur e 4: T he anal ysis of teeth in an adult fr og. (A) The fr ontal vie w of an adult female fr og skull is depicted with an upper maxillary arc h with maxillary teeth visualized in y ello w. (B) The lateral and top view on the right half of the maxillary arch with visualized teeth from an adult female frog. Thr ee differ ent de v elopmental sta ges of teeth ar e highlighted by differ ent colors (y ello w, c y an, orange). (C) The stained micro-CT scan shows that only a small portion of the tooth extends into the oral ca vity. T he arrows point to teeth rows that do not penetrate the oral ca vity. (D) T he later al vie w of the mandible of an adult female frog confirms the absence of teeth in this area. (E) The lateral view of the rostral part of the maxilla displays different stages of teeth during the replacement of tooth rows in detail. refer to Supplementary Fig. S3 to see how the analysis was performed). Based on our micr o-CT anal ysis, in terms of sex, we found that adult females can be viewed as essentiall y enlar ged males, indicating a proportional growth pattern (Fig. 5 C). We also investigated whether the de v elopment of the left and right sides of X. laevis is uniform or div er gent ( Supplementary Fig. S4 ), and consistentl y, we observ ed L-R length symmetry in the length of all long bones . T her efor e, we next focused our detailed examination only on one side of the fr og, specificall y the left side (L). Our findings r e v ealed that certain limb bones, particularly those in the hindlegs, such as the femur and tibiofibular, exhibited r elativ el y r a pid gr owth, while other bones suc h as for elimb ones gr e w at a slo w er r ate (Fig. 5 C). Importantl y, this differ ence was not attributed to absolute bone measurements but rather in relation to the animal’s overall length (data not shown). This phenomenon may be linked to the frogs’ utilization of their hindlimbs for swimming, wher eas e v en their for elimbs ar e primaril y emplo y ed for food handling [ 62 , 63 ]. Mor eov er, the length of the tarsal bone, such as astragalus and calcaneum, appears to be less critical for escape or startle r esponses, suc h as swim kicking, compared to the lengths of the femur and tibiofibular, whic h ar e correlated with the muscle mass of the thigh and the calf [ 7 ]. It is also Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024 8 | GigaScience , 2024, Vol. 13 Figur e 5: T he anal ysis of fr og skeletogenesis . (A) T he adult male fr og with anal yzed bones is depicted. (B) The adult female fr og with anal yzed bones is displa yed. (C) T he gr a ph demonstr ating the anal yzed bones thr oughout the Xenopus laevis ’ late de v elopment. T he experiment in volved conducting 3 repetitions for each long bone measurement, with the results depicted as means accompanied by standard deviations ( ±SD). Howe v er, err or bars are depicted only for cases demonstrating statistically significant differences, while they are not displayed for insignificant variations. Statistical analysis was performed using a 2-way analysis of variance (ANOVA) follo w ed b y Tuk e y post hoc test for multiple comparisons; ∗∗∗∗P < 0.0001. A: anterior; AS: astr a galus; CA: calcaneum; F: femur; H: humerus; L: left; MC: metacarpals; MT: metatarsals; P: posterior; R: right; RU: radioulna; TF: tibiofibular. Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024 Unv eiling v ertebr ate de v elopment dynamics in fr og | 9 intriguing to note that most bones initiated their growth from r oughl y the same size but terminated it at different dimensions . For example , metatarsals de v elop at a faster rate than metacarpals (Fig. 5 C), which aligns with functional considerations, as outlined else wher e [ 37 ]. Together, based on our micr oCT data, one can make new predictions for further experimental testing of (long) bone growth. Ossification Analysis Besides bone length, it is also feasible to analyze the bone mass using our Xenopus dataset. Within the frog skeleton, 2 distinct types of bones can be gener all y distinguished in terms of their de v elopmental origin. Dermal ossification, originating in the dermis, is evident in certain skull elements and in 2 bones of the pector al girdle, namel y, the cleithrum and cla vicle . Con v ersel y, the remaining components of the postcranial skeleton consist of either cartilage or bones replacing the cartila ge, ac hie v ed thr ough endoc hondr al ossification [ 19 ]. As the bones of the limbs undergo development in both tadpoles and adults, they typically comprise an ossified section alongside cartila ge. Notabl y, the ossification pr ocess al ways commences at the central regions of long bones, as shown on the example of the femur (Fig. 6 A). By assessment of the mass ratio between cartilage and bone (Fig. 6 B–B  ; Supplementary Fig. S5 ), we observed that, with a few exceptions, all long bones share relativ el y similar pr oportions (data not shown). Furthermor e, in both adult males and females, there is a lack of dynamic growth akin to the de v elopmental sta ges, with onl y a noticeable incr ease in mass (Fig. 6 C). After climax metamorphosis, no proliferative ossification occurs, and the process is limited to the calcification of cartila ge, irr espectiv e of the fr og’s sex (Fig. 6 C). Mor eov er, the r elativ e r atio of cartila ge to an ossified bone diminishes as de v elopment pr ogr esses, exemplified by the fem ur, humerus, and r adioulna (Fig. 6 C, D). T hus , r esearc hers can further tak e ad v anta ge of these indications to compare the growth of different bones in v arious a ged animals. Segmentation of Selected Internal Soft Organs Our dataset offers significant potential not only for the e v aluation of hard bones but also for soft tissue such as internal organs. Precise segmentation of structures is essential because once the structure in focus is clearly distinguished, further assessment of its morphology and intraspecies differences becomes considerably easier and more accurate . T his tool also provides the flexibility to describe each internal structure in detail either separately or in the context of its individual surrounding elements. In addition to studying differences during X. laevis ’ development, segmented structures can be utilized (e.g., for investigating intersex differences). For an illustration of the possible application of our micr o-CT data, her e we further selected 2 k e y v ertebr ate or gans, such as the brain and gut. T he br ain It is noteworthy that our micro-CT data allow for detailed observation of the developmental segmentation of various regions of the br ain, whic h is v ery hard to dissect, especiall y fr om Xenopus adults (personal observ ation). Specificall y, Fig. 7 A–E, A  –E  , and Supplementary Video S4 provide a clear morphological visualization of the individual brain areas such as cerebral hemispheres (cbh), cerebellum (cbl), diencephalon (dch), medulla oblongata (mob), optic lobes (opl), and spinal cord (sp), in the tadpole and adult frog brains, as well as to follow how these structures are developing in time course. For more information about the X. laevis ’ brain and its detailed description, including its r egener ation in developing tadpoles, we refer readers to the recent publication [ 34 ]. Subsequently, we also asked whether we could analyze not only the brain but also the brain-associated nerves like nervus opticus using the micro-CT dataset (Fig. 7 F). Indeed, we could see that the nerv es attac hed to ey es slo wly elongated and then modified, as a specimen size changed during development (Fig. 7 F). Together, the quality of our micro-CT allows us to investigate not only the individual brain parts but also its associated structures. The gut During the metamorphosis of X. laevis , the gut undergoes significant remodeling, with the intestine shortening by a ppr oximatel y 75% over an 8-day period. The coiling pattern changes, with the outer loops initially coiling counterclockwise and the inner loops coiling cloc kwise, r e v ersing at the ileum’s switc hbac k point [ 64 , 65 ]. This remodeling is characterized by a stage-dependent sequential organization of nascent smooth muscle cells, which plays a crucial role in gut coiling morphogenesis [ 66 ]. To our best knowledge, this process has not been studied using micro-CT so far. T hus , we selected the gut as a second internal organ to dissect by the micro-CT technique, with a focus on the whole X. laevis atlas (Fig. 8 ). It is evident from the micro-CT images that it successfully depicted the gut structure with high quality and detail (Fig. 8 and Supplementary Video S5 ). For more information about the X. laevis gut and its anatomy in general, we r efer a r eader to the Xenopus illustrations by Zahn and colleagues [ 52 ] and supporting liter atur e [ 64 , 65 ]. In sum, r esearc hers will be able to use our micro-CT dataset to segment and explore several internal organs, including the brain or gut, as well as associated structur es suc h as nerv es, for further analytical and practical purposes, as mentioned in Discussion below. Discussion This study used micro-CT to create high-resolution 3D models of X. laevis from tadpoles to adults, revealing detailed dev elopmental and mor phological c hanges . T he micro-CT scan repository enhances understanding of X. laevis ’ de v elopment and has applications in research, virtual reality, 3D printing, and education. The X. laevis ’ de v elopment dataset not only offers valuable insights into this amphibian species but also presents a wealth of biological potential as it serves as a starting point for comparativ e, r egener ativ e, and e volutionary biology as well as morphological study. The dataset’s diverse specimen representation and the high quality of the scans open up a realm of possibilities for advanced subsequent analyses. One intriguing avenue of study lies in the examination of hard tissue growth. The dataset provides an ideal platform to inv estigate especiall y the long bones of Xenopus but also v ertebr ae, or a skull. Furthermor e, the dataset facilitates the analysis of teeth or cartilage, which we have addressed using dedicated tools in VG Studio Max. Next, this dataset lends itself to in-depth exploration by researc hers inter ested in the anatomy of Xenopus , including internal organs . 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Methods Mol Biol 2023;2633:111–30. https://doi.org/ 10.1007/9781071630044 _ 10 . Recei v ed: November 30, 2023. Revised: April 10, 2024. Accepted: June 3, 2024 ©The Author(s) 2024. Published by Oxford Uni v ersity Pr ess on behalf of GigaScience. This is an Open Access article distributed under the terms of the Cr eati v e Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giae037/7714386 by Technical University of Brno user on 23 July 2024