Transcriptomic analysis of intermuscular bone development in barbel steed (Hemibarbus labeo)

Intermuscular bones (IBs), which are little, bony spicules in muscle, are embedded in lower teleosts' myosepta. Despite the importance of studying IB development in freshwater aquaculture species, the genes associated with IB development need to be further explored. In the present study, we identified four stages of IB development in barbel steed (Hemibarbus labeo), namely stage 1: IBs have not emerged, stage 2: a few small IBs have emerged in the tail, stage 3: longer IBs gradually emerged in the tail and stage 4: all of the IBs in the tail are mature and long, via Alizarin red staining. Subsequently, we used the HiseqXTen platform to sequence and de novo assemble the transcriptome of epaxial muscle (between 35th and 40th myomere) of barbel steed at 29 days (stage 1) and 42 days (stage 3) after hatching. A total of 190,814 unigenes were obtained with an average length and N50 of 648 bp and 1027 bp, respectively. We found 2174 differentially expressed genes (DEGs) between stages 1 and 3, of which 378 and 1796 were up- and down-regulated, respectively. Functional enrichment analysis showed that several DEGs functioned in ossification, positive regulation of osteoblast differentiation, osteoblast differentiation, and BMP signaling pathway, and were further enriched in signal pathway, including osteoclast differentiation, TGF-β signaling pathway, cytokine-cytokine receptor interaction, Jak-STAT signaling pathway, and other KEEG pathways. In conclusion, we identified genes that may be related to IB development, such as kazal type serine peptidase inhibitor domain 1 (KAZALD1), extracellular matrix protein 1 (ECM1), tetranectin, bone morphogenetic protein 1 (bmp1), acid phosphatase 5 (ACP5), collagen type XI alpha 1 chain (COL11A1), matrix metallopeptidase 9 (MMP9), pannexin-3 (PANX3), sp7 transcription factor (Sp7), and c-x-c motif chemokine ligand 8 (CXCL8), by comparing the transcriptomes of epaxial muscle before and after IB ossification. This study provided a theoretical basis for identifying the molecular mechanisms underlying IB development in fish.