Integrated transcriptomic analysis reveals evolutionary and developmental characteristics of tendon ossification in teleost

Background Intermuscular bones (IBs) are segmental ossifications in the myosepta and generally are similar with tendon ossification which is a type of heterotopic ossification (HO). Nevertheless, mechanism of IB formation is not well understood. Results In order to address this issue, we systematically analyze transcriptome data across multiple tissues, multiple species, multiple time points and multiple resolutions in teleost. First of all, we identify IB-specific genes by tau index method. Through cross-species comparison of tendon development process, we discover that candidate genes primarily are enriched in extracellular matrix organization, ossification, regulation of angiogenesis, et al. We also reveal that some of above candidate genes were abnormally expressed in runx2b ^−/− zebrafish without IBs. With the hope of clarifying the trajectory of cell differentiation during IB formation, we show that myostptal stem cell differentiates into osteoblast, fibroblast and tenocyte in runx2b ^+/+ zebrafish. However, the differentiation process of myostptal stem cell into osteoblast is inhibited in runx2b ^−/− zebrafish, while the differentiation process of myostptal stem cell into clec3bb  + tenocyte and fibroblast is enhanced. Meanwhile, runx2b deficiency led to the upregulation of clec3bb expression in clec3bb  + tenocyte cluster. Notably, there was a compensatory effect observed in cell differentiation and gene expression in runx2b ^−/− zebrafish, which suggests that runx2b and these candidate genes (such as clec3bb ) are involved in the gene network of IB development. Conclusions This study elucidated the cell development process of tendon ossification in teleost, and identified key factor clec3bb involved in tendon ossification. These findings provide a foundation for understanding tendon ossification in teleost and further research on tendon ossification in mammals.