关键词: 昼夜节律, BMAL1, CLOCK, 免疫组织化学, RT-PCR, 心脏, 肝脏, 骨骼肌, 胚胎, 组织建构

Abstract:

BACKGROUND: Expression and mechanisms of periodic rhythm emergence during mammalian development are not fully understood.
OBJECTIVE: To study the spatio-temporal expression characteristics of clock gene in mouse embryos at the gestational age, maternal livers, and maternal skeletal muscles during the development of the mouse. 
METHODS: The whole embryos from pregnant 10-17-day ICR mice were collected. The mouse heart, lung, liver and skeletal muscle of body underwent immunohistochemical staining by anti-CLOCK and -Per1 antibodies. Total RNA was extracted using TRIZol from embryos, quadriceps and liver. BMAL1, CLOCK, Per1, myogenin, Tcap and MAZ mRNA were examined by RT-qPCR.
RESULTS AND CONCLUSION: The levels of BMAL1, CLOCK and Per1 expression in mouse embryo in the early developmental stages were lower. Immunohistochemical analysis showed that the heart of the early embryos did not express CLOCK at gestational days 10 and 11. However, apparent signals against Per1 and weak expression of CLOCK were observed in the maternal uterus tissue (decidua) of embryos at gestational days 10 and 11. At gestational day 13, CLOCK positive cells showed C-shaped patterns in the wall of the left and right bronchi. At the same time, CLOCK positive cells were detected in the bottom of left atrium and Per1 positive cells were detected in the left ventricular myocardium. At gestational day 14, CLOCK and Per1 positive cells were easily observed in skeletal muscle of body wall. At gestational day 15, immunohistochemical analysis showed a small number of Per1 positive cells were observed in the bottom of right atrium. The cross section of bronchial branches in the lungs increased obviously. At the same time, the expression levels of BMAL1, CLOCK and Per1 mRNA in mouse embryo were increased, especially Per1. However, circadian molecular rhythms could not be found. We found the level of Tcap expression at gestational day 15 significantly increased. The data suggest that sarcomeres in muscle develop rapidly. During development from gestational day 16 to gestational day 17, CLOCK and Per1 positive cells were also observed in the airway smooth muscle, the wall of left atrium and skeletal muscle of body. During the development, CLOCK and Per1 positive cells could not be seen in the liver. The level of myogenin expression in mouse embryo in the early developmental stages was lower. The level of MAZ expression is the highest than myogenin and Tcap in embryos. MAZ may concern with the development and differentiation of skeletal muscle. It is indicated that circadian clock development in embryos is closely correlated with the cellar differentiation process. Mouse embryos begin to express key circadian genes and have the capacity to express active circadian regulatory cycles during development. The clock gene is a positive regulator of myogenesis and the development of heart and liver. The right ventricle shows a relatively slow pace of maturation. However, the present results indicate that synchrony does not occur during prenatal development despite exposure to maternal rhythms. It may be affected by maternal skeletal muscles of mice.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Genes, Biological Clocks, Muscle, Skeletal, Embryology, Tissue Engineering