The Role Of MsxC Gene In The Development Of Axial Muscle And Intermuscular Bones In Zebrafish

Intermuscular bones,are located in the muscle space on both sides of the vertebrae and are formed by the ossification of the connective tissue of the muscle septum.The formation of intermuscular bones may be closely related to muscle development.The order of formation of intermuscular bones is different for fishes of different swimming styles.Among the fish swimming in the Sub-carangiform type,such as silve carp,bighead carp,and Megalobrama amblycephala,the intermuscular ossicles are ossified from the tail to the front,while the Anguilliform type swimming fish is reversed.The Japanese eel muscles are ossified from the head to the tail.It is suggested that the formation of intermuscular ossicles is induced by the mechanical force generated by muscle swing during swimming,so the formation of thrust intermuscular bones might be related to the muscle strength of the large lateral muscles.MsxC?muscle segment homeobox 3,msx-c,msx3,et ID309953.13,msh-C,msh C,zgc:86596?genes belong to the Hox gene family,bone morphogenic protein?BMP?,fibroblasts Fibroblast growth factor?FGF?and its receptors?FGF receptor ?FGFR?,Dlx?distallessgene?,transforming growth factor?TGF?,etc.?participate in the epithelial-mesenchymal interaction.Osteocytes are differentiated from mesenchymal cells during the process of interstitial myogenesis.MsxC can be an important candidate gene to promote the differentiation of osteoblast precursors of intermuscular ossicles.In addition,it was found in the lip fish Hemibarbus labeo that the expression level of MsxC was the highest before the formation of the intermuscular bones,especially the time of its expression in the axial muscle and the intermuscular septum,which was consistent with the formation process of the intermuscular bones.It is speculated that MsxC may be related to the occurrence of intermuscular bones.As a model organism,zebrafish?Danio rerio?has been widely used in genetics,developmental biology,and other researches.The CRISPR/Cas9 gene editing technology is used to knock out the zebrafish MsxC gene to obtain the corresponding mutant strain.It was found that there were fewer muscle cells in the muscle compartment of the mutant zebrafish adult after knocking out the MsxC gene,especially in the area around intermuscular septum,indicating that the knockout of the gene did affect the arrangement of the muscle compartment.Immunohistochemical tests showed that there were more firble of red muscle in MsxC^-/-mutant than that in wild strain of zebrafish.Meanwhile,we also found that the intermuscular bones of this mutant became shorter and thinner than the wild type,and the area of ossification decreased,indicating that the formation of intermuscular spines is related to the growth and development of the muscles of the lateral muscles.In addition,we also tested the effect of MsxC on bone formation during embryonic development,and found that head cartilage was dysplastic at three day after fertilization and adult fish's head bone developed abnormally in MsxC^-/-mutant,further indicating that the MsxC gene plays roles in the development of craniofacial bone.In this study,genome editing technology CRISPR/Cas9 was used to creat the MsxC^-/-mutant.Both the growth of axial muscle and the formation of intermuscule bones were found to be affected in the mutant,providing a new data to better understand the developmental mechanism of intermuscular bones.In addition,compared to previous reports in the zebrafish using Morpholino?MO?to knock down the MsxC gene to produce a phenotype of craniofacial cartilage changes,the knockout of this gene in the study produced a new phenotype:an increase in Merkel's cartilage area,follow-up to adult fish with stunted Merkel cartilage,dental bones and some other bones.The establishment of the knockout model lays the foundation for further exploring the function of MsxC gene.