BACKGROUND:Mouse, 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.RESULTS:The 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.CONCLUSION:These 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.

AbstractScaffolds play a key role in bone tissue engineering (BTE) as they provide a mechanically and biologically supportive template to treat bone defects. Yet, the ideal scaffold structures are far from certain, leaving a lot to be discovered in terms of the scaffold structure–performance relationships. In this study, we investigated the influence of pore size and internal structure on osteoblast performance in vitro. Three‐dimensional (3D) scaffolds were printed from polycaprolactone (PCL) reinforced with 30% (wt.) nano‐hydroxyapatite (nHAp), with two different internal structures (lattice and staggered) and four pore sizes (0.280, 0.380, 0.420, and 0.550 mm). Scaffolds were seeded with pre‐osteoblast cells (MC3T3‐E1). Metabolic activity of cells, osteoblast differentiation, and capability of osteoblasts to deposit mineralized matrix were examined in vitro. Staggered scaffolds better supported cellular performance. The pore size of 0.280 mm was more favorable to support cell proliferation while the pore size ≥0.420 mm was more effective to promote osteoblast differentiation and mineralization. Findings revealed that osteoblast activities were affected differently by the pore size. Our study further suggests that the structure with a gradient pore size would be better than a single size in terms of supporting cell proliferation, differentiation, and secretion of a mineralized matrix.

To explain how cartilage appeared in different parts of the vertebrate body at discrete times during evolution, we hypothesize that different embryonic populations co-opted expression of a core gene regulatory network (GRN) driving chondrocyte differentiation. To test this hypothesis, laser-capture microdissection coupled with RNA-seq was used to reveal chondrocyte transcriptomes in the developing chick humerus and ceratobranchial, which are mesoderm- and neural crest-derived, respectively. During endochondral ossification, two general types of chondrocytes differentiate. Immature chondrocytes (IMM) represent the early stages of cartilage differentiation, while mature chondrocytes (MAT) undergo additional stages of differentiation, including hypertrophy and stimulating matrix mineralization and degradation. Venn diagram analyses generally revealed a high degree of conservation between chondrocyte transcriptomes of the limb and head, including SOX9, COL2A1, and ACAN expression. Typical maturation genes, such as COL10A1, IBSP, and SPP1, were upregulated in MAT compared to IMM in both limb and head chondrocytes. Gene co-expression network (GCN) analyses of limb and head chondrocyte transcriptomes estimated the core GRN governing cartilage differentiation. Two discrete portions of the GCN contained genes that were differentially expressed in limb or head chondrocytes, but these genes were enriched for biological processes related to limb/forelimb morphogenesis or neural crest-dependent processes, respectively, perhaps simply reflecting the embryonic origin of the cells. A core GRN driving cartilage differentiation in limb and head was revealed that included typical chondrocyte differentiation and maturation markers, as well as putative novel “chondrocyte” genes. Conservation of a core transcriptional program during chondrocyte differentiation in both the limb and head suggest that the same core GRN was co-opted when cartilage appeared in different regions of the skeleton during vertebrate evolution.

Vertebrates are the only animals that produce bone, but the molecular genetic basis for this evolutionary novelty remains obscure. Here, we synthesize information from traditional evolutionary and modern molecular genetic studies in order to generate a working hypothesis on the evolution of the gene regulatory network (GRN) underlying bone formation. Since transcription factors are often core components of GRNs (i.e., kernels), we focus our analyses on Sox9 and Runx2. Our argument centers on three skeletal tissues that comprise the majority of the vertebrate skeleton: immature cartilage, mature cartilage, and bone. Immature cartilage is produced during early stages of cartilage differentiation and can persist into adulthood, whereas mature cartilage undergoes additional stages of differentiation, including hypertrophy and mineralization. Functionally, histologically, and embryologically, these three skeletal tissues are very similar, yet unique, suggesting that one might have evolved from another. Traditional studies of the fossil record, comparative anatomy and embryology demonstrate clearly that immature cartilage evolved before mature cartilage or bone. Modern molecular approaches show that the GRNs regulating differentiation of these three skeletal cell fates are similar, yet unique, just like the functional and histological features of the tissues themselves. Intriguingly, the Sox9 GRN driving cartilage formation appears to be dominant to the Runx2 GRN of bone. Emphasizing an embryological and evolutionary transcriptomic view, we hypothesize that the Runx2 GRN underlying bone formation was co-opted from mature cartilage. We discuss how modern molecular genetic experiments, such as comparative transcriptomics, can test this hypothesis directly, meanwhile permitting levels of constraint and adaptation to be evaluated quantitatively. Therefore, comparative transcriptomics may revolutionize understanding of not only the clade-specific evolution of skeletal cells, but also the generation of evolutionary novelties, providing a modern paradigm for the evolutionary process.