Common features of cartilage maturation are not conserved in an amphibian model

Abstract

BackgroundMouse, chick, and zebrafish undergo a highly conserved program of cartilage maturation during endochondral ossification (bone formation via a cartilage template). Standard histological and molecular features of cartilage maturation are chondrocyte hypertrophy, downregulation of the chondrogenic markers Sox9 and Col2a1, and upregulation of Col10a1. We tested whether cartilage maturation is conserved in an amphibian, the western clawed frog Xenopus tropicalis, using in situ hybridization for standard markers and a novel laser‐capture microdissection RNAseq data set. We also functionally tested whether thyroid hormone drives cartilage maturation in X tropicalis, as it does in other vertebrates.ResultsThe developing frog humerus mostly followed the standard progression of cartilage maturation. Chondrocytes gradually became hypertrophic as col2a1 and sox9 were eventually down‐regulated, but col10a1 was not up‐regulated. However, the expression levels of several genes associated with the early formation of cartilage, such as acan, sox5, and col9a2, remained highly expressed even as humeral chondrocytes matured. Greater deviances were observed in head cartilages, including the ceratohyal, which underwent hypertrophy within hours of becoming cartilaginous, maintained relatively high levels of col2a1 and sox9, and lacked col10a1 expression. Interestingly, treating frog larvae with thyroid hormone antagonists did not specifically reduce head cartilage hypertrophy, resulting rather in a global developmental delay.ConclusionThese data reveal that basic cartilage maturation features in the head, and to a lesser extent in the limb, are not conserved in X tropicalis. Future work revealing how frogs deviate from the standard cartilage maturation program might shed light on both evolutionary and health studies.

Proteoglycan inhibition of canonical BMP-dependent cartilage maturation delays endochondral ossification

“…To evaluate whether BMP signalling was active in developing cartilage of zebrafish, laser capture microdissection of cranial cartilage was performed, avoiding perichondral cells, followed by RNA sequencing ( Gomez-Picos et al, 2022 ; Nguyen et al, 2023 ). Several members of the BMP signalling pathway were expressed in cranial cartilage of 6 days post-fertilization (dpf) wild-type zebrafish larvae ( Table S1 ).…”

“…RNA was isolated using laser capture microdissection of tissue sections of cranial cartilage in the occipital region of wild-type 6 dpf zebrafish larvae, amplified, and sequenced as described previously ( Gomez-Picos et al, 2022 ; Nguyen et al, 2023 ). Three independent samples were processed, and normalized read counts were determined as described previously ( Gomez-Picos et al, 2022 ; Nguyen et al, 2023 ).…”