Gene expression in extant animals might reveal how skeletal cells have evolved over the past 500 million years. The cells that make up cartilage (chondrocytes) and bone (osteoblasts) express many of the same genes, but they also have important molecular differences that allow us to distinguish them as separate cell types. For example, traditional studies of later‐diverged vertebrates, such as mouse and chick, defined the genes Col2a1 and sex‐determining region Y‐box 9 as cartilage‐specific. However, recent studies have shown that osteoblasts of earlier‐diverged vertebrates, such as frog, gar, and zebrafish, express these ‘chondrogenic’ markers. In this review, we examine the resulting hypothesis that chondrogenic gene expression became repressed in osteoblasts over evolutionary time. The amphibian is an underexplored skeletal model that is uniquely positioned to address this hypothesis, especially given that it diverged when life transitioned from water to land. Given the relationship between phylogeny and ontogeny, a novel discovery for skeletal cell evolution might bolster our understanding of skeletal cell development.

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.