Loss of scleraxis leads to distinct reduction of mineralized intermuscular bone in zebrafish

Intermuscular bones (IBs) are ossified from tendons and only occur in lower teleosts. Positive association between the regulation of scleraxis gene (scx) and tendon development gave us reasons to speculate that the scx gene may play a potential role in regulating the development of IBs. A phylogenetic analysis conducted for this study revealed potential functional differentiation between two scx orthologues, scxa and scxb. The scxa−/− and scxb−/− zebrafish were generated through CRISPR-Cas9 technology to study the role of scx in the IB and rib development. The results showed a significant reduction of the number of IBs in adult scxa-1-/- zebrafish, with almost 70% reduction (15–25 IBs) compared to the wild type scxa-1+/+ zebrafish (76–80 IBs). In the scxa-1+/+ adults, IBs were observed in both dorsal and tail segments; however, in scxa-1-/- fish IBs were observed only in the tail segment (none in the dorsal segment). Although scxa-1−/− zebrafish had rib defects, the mutants were viable and fertile as adult fish. The scxb−/− zebrafish had the same number of IBs and same skeletal phenotype as the wild-type fish. This suggests that only scxa has a crucial role in the IB development, and confirms functional differentiation of scx orthologues. To further clarify the molecular mechanism by which scxa affects the IB development, we conducted comparative transcriptome analysis of dorsal tissue samples of scxa-1+/+ (with IBs) and scxa-1−/− (without IBs), and further verified the expression of key genes via qPCR. This is the first study to identify a gene that controls the amount of IBs in fish, and it provides a new sight into the effects of scxa on the molecular mechanism of IB development in fish.