Abstract: BACKGROUND: Skeletal muscle has a circadian rhythm, but the mechanism of circadian Clock genes during the development and differentiation of the skeletal muscle and the interrelationship with muscle regulators are not fully understood.
OBJECTIVE: To observe the spatio-temporal expression characteristics of circadian Clock genes in embryos of different mouse embryonic ages and during the differentiation of C2C12 myoblasts as well as to examine the relationship of Bmal1, Clock and MyoD in the skeletal muscle development.
METHODS: RT-qPCR was used to examine Bmal1, Clock, Per1, Cry1, Rev-erbα, as well as myogenic regulatory factors MyoD, myogenin, Tcap and MAZ from the embryos of ICR mice at embryonic ages of 10-17 days and during the differentiation of C2C12 myoblasts. Western blot was used to investigate the expression of Bmal1, Clock and MyoD at 48, 96, and 144 hours after differentiation of C2C12 myoblasts. Luciferase reporter assay was used to analyze the activation of TCAP reporter plasmids by co-transfection of Bmal1, Clock and MyoD plasmids. An approval for this study was obtained from the Animal Experimental Ethics Committee of Shanxi Medical University. 
RESULTS AND CONCLUSION: (1) With the development of mouse embryo, the expression of Bmal1, Clock, and Per1 were increased, especially Per1. Cry1 and Rev-erbα were weakly expressed. (2) The expression intensity of MyoD and myogenin were gradually increased. In addition, the level of Tcap expression was related to sarcomere development. The expression of MAZ maintained a higher level and sustained increased in embryos, which might be involved in the development and differentiation of the skeletal muscle. (3) With the induced differentiation of C2C12 myoblasts, the expressions of Bmal1 and Clock were synchronously increased. At the same time, the expression of Cry1 was up-regulated gradually. Per1 and Rev-erbα were very weakly expressed. The expressions of MyoD and myogenin were gradually increased, specially myogenin. This indicates that C2C12 myoblasts are the optimal model for research on myogenic differentiation. Western blot analysis revealed that the protein levels of Bmal1 and Clock were highly expressed in C2C12 myoblasts, which might be necessary for remaining the activity of myogenic regulatory factor. MyoD was highly expressed, but the band of MyoD  did not achieve statistical significance. By the luciferase reporter assay, we found that Bmal1, Clock and MyoD  plasmids worked in synergistic fashion to regulate the expression of TCAP (Titin-cap). To conclude, Bmal1, Clock and MyoD are all key regulators of myogenic differentiation. Highly expressed MyoD may be related to phase separation and play an important role in the development and differentiation of skeletal muscle. Persistently expressed Bmal1 is beneficial to maintain the activity of muscle regulators. Lowly expressed Rev-erbα is beneficial to skeletal muscle maturation. Per1, which is consistently highly expressed during embryonic development, may be involved in the establishment of the inhibition of cyclic rhythms and is beneficial to the development of the skeletal muscle and other tissues and organs.

Key words: skeletal muscle, embryos, circadian Clock gene, mouse, C2C12