Single-cell transcriptomes and runx2b-/- mutants reveal the genetic signatures of intermuscular bone formation in zebrafish

Intermuscular bones (IBs) are mineralized spicules, present in the myosepta of many, but not all, teleost species. IBs are often small and sharp, and they consequently limit how the fish can be processed; the IBs may cause injury or trauma if lodged in consumers' throats or mouths, and therefore affect the appeal of the fish to many consumers. The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established. Here, the characteristics of IB tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish. The analysis defined 18 distinct cell types. Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b(-/-) mutants completely lost IBs, while swimming performance, growth and bone mineral density were not significantly different from runx2b(+/+) zebrafish. Comparative single-cell RNA sequencing (scRNA-seq) analysis in runx2b(-/-) and runx2b(+/+) zebrafish revealed the role of osteoblasts in IB formation. In addition, differentially expressed genes were enriched in the transforming growth factor beta/bone morphogenetic protein (TGF-beta/BMP) pathway after runx2b deletion. This study provides evidence for the crucial role of runx2b regulation in IB formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs, which can improve the safe consumption and economic value of many farmed fish species.

Automated summary

IBs are mineralized spicules found in the myosepta of some teleost species. This study evaluated the characteristics of IB tissue using single-cell transcriptomics in zebrafish and identified 18 distinct cell types. The analysis showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. The researchers also identified 10 candidate genes and demonstrated the crucial role of runx2b regulation in IB formation. This research provides a genetic breeding strategy to generate commercial fish species without IBs, improving their safety and economic value.