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Dev Growth Differ
2023 Jan 01;651:16-22. doi: 10.1111/dgd.12831.
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Diversity of cortical bone morphology in anuran amphibians.
Kondo Y
,
Iwamoto R
,
Takahashi T
,
Suganuma K
,
Kato H
,
Nakamura H
,
Yukita A
.
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The cortical bones of mammals, birds, and reptiles are composed of a complex of woven bone and lamellar bone (fibrolamellar bone) organized into a variety of different patterns; however, it remains unclear whether amphibians possess similar structures. Importantly, to understand the evolutionary process of limb bones in tetrapods, it is necessary to compare the bone structure of amphibians (aquatic to terrestrial) with that of amniotes (mostly terrestrial). Therefore, this study compared the cortical bones in the long bones of several frog species before and after metamorphosis. Using micro-computed tomography (CT), we found that the cortical bones in the fibrolamellar bone of Xenopus tropicalis (Pipoidea superfamily) and Lithobates catesbeianus (Ranoidea superfamily) froglets are dense, whereas those of Ceratophrys cranwelli (Hyloidea superfamily) are porous. To clarify whether these features are common to their superfamily or sister group, four other frog species were examined. Histochemical analyses revealed porous cortical bones in C. ornata and Lepidobatrachus laevis (belonging to the same family, Ceratophryidae, as C. cranwelli). However, the cortical bones of Dryophytes japonicus (Hylidae, a sister group of Ceratophryidae in the Hyloidea superfamily), Microhyla okinavensis (Microhylidae, independent of the Hyloidea superfamily), and Pleurodeles waltl, a newt as an outgroup of anurans, are dense with no observed cavities. Our findings demonstrate that at least three members of the Ceratophryidae family have porous cortical bones similar to those of reptiles, birds, and mammals, suggesting that the process of fibrolamellar bone formation arose evolutionarily in amphibians and is conserved in the common ancestor of amniotes.
Figure 1. Images of a representative micro-CT reconstruction of the frogs' tibiofibula. (a and b) Xenopus tropicalis, (c) Lithobates catesbeianus, and (d) Ceratophrys cranwelli. Sagittal section (a) and transverse sections (b–d) of the tibiofibula are shown. All images are surface-rendered 3D-reconstructed images. The insets at the upper right of (b–d) are the respective tomographic images. Arrows: Cavities observed in cortical bone. All frogs shown in this figure are young individuals within a few weeks of completing metamorphosis.
Figure 2. Cortical bone formation in Xenopus tropicalis. (a–d) Mid-diaphyseal cross-sections of the X. tropicalis tibiofibula. (a) Tadpoles undergoing metamorphosis with limb formation in progress (Gosner stage 37/NF stage 57). (b) Tadpoles that are completing metamorphosis into frogs but still have their tails (Gosner stage 42/NF stage 60). (c) Young frog just after completion of metamorphosis (Gosner stage 46/NF stage 66). (d) Adult frog (14-months old). (a'–d') higher magnifications of the squares in (a–d), respectively. Arrows: Osteocytes, arrowheads: Osteoblasts. BM, bone marrow; Ca, cartilage; CB, cortical bone; pe, periosteum. Scale bars: 1 mm.
Figure 3. Cortical bone formation in Ceratophrys cranwelli. (a–d) Mid-diaphyseal cross-sections of the C. cranwelli tibiofibula. (a) a tadpole that has begun metamorphosis, with its hind limbs starting to form, but its skin still larval (Gosner stage 37). (b) a tadpole which has undergone further metamorphosis into a frog, and its skin has changed to adult form; however, it still retains its tail (Gosner stage 42). (c) a young frog just after completion of metamorphosis (Gosner stage 46). (a'–c') higher magnifications of the squares in (a–c), respectively. (d) Mature frog (12–months old). Porous bone was observed in the area where the tibia and femur were fused (dashed area). Arrowheads: Osteoblasts; black arrows: Osteocytes; white arrows: Blood vessels; asterisk: Osteoclast. BM, bone marrow; Ca, cartilage; CB, cortical bone; pe, periosteum. Scale bars in (a–c) 1 mm, in (d) 1 cm.
Figure 4. Histological analysis of long bones of Ceratophryidae, Dryophytes japonicus and Microhyla okinavensis. Transverse sections of C. ornata (a) and L. laevis (b) in the mid-diaphyseal region of the tibiofibula and transverse sections of D. japonicus (C) and M. okinavensis (d) in the mid-diaphyseal region of the femur are shown. C. ornata and L. laevis shown in this figure were young frogs, a few weeks after metamorphosis (in length 3–4 cm). D. japonicus and M. okinavensis shown in this figure were froglets that had just undergone metamorphosis. Scale bars in a: 5 mm, in (b–d) 1 mm.
Fig. S1 Photographs of X. tropicalisand C. cranwelliused in Figures 2 and 3. These frogs were staged according to Gosner'stable (Gosner, 1960). (A-D): X. tropicalis, (E-H): C. cranwelli. One square in the background represents a 1 cm2square.
Fig. S2 Histological analysis of femur of Ceratophyridae. Sagittal sections of femur of C. cranwelli(A), C. ornata (B) and L. laevis(C) are shown. (A’–C’) Higher magnifications of the squares in (A), (B), and (C), respectively. All frogs shown in this figure were young frogs those had passed a few weeks after metamorphosis (in length 3-4 cm).
Fig. S3 Histological analysis of femur of L. catesbeianus. Transversesections of femur of young (A) and mature (B) frogs are shown. (A’ and B’) Higher magnifications of the squares in (A) and (B), respectively.
Fig. S4 Histological analysis of long bones of X. laevis. Sagittalsections of tibiofibula of NF-stage 59 (A) and young (B) frogs are shown. (A’ and B’) Higher magnifications of the squares in (A) and (B), respectively.
Fig. S5 Histological analysis of femur of P. waltl. Sagittalsections of femur of immature (A) and young (B) P. waltlare shown. (A’ and B’) Higher magnifications of the squares in (A) and (B), respectively.
