Temporal expression of myogenic regulatory genes during embryonic development
During the embryonic development of both males and females, the expression levels of the Myomaker gene in pectoral and thigh muscles among the four chicken breeds showed a wave change trend of first increasing and then decreasing (Fig. 2A-D). The Myomaker mRNA abundance in both muscle tissues showed an overall increase from E11 to E15 and reached the highest level at E13 of BYC (Fig. 2B) and at E15 (Fig. 2A) of the other three breeds (Fig. 2C-D), which means that the critical period of myogenic fusion in chicken embryos was from E13 to E15. Afterward, the expression of the Myomaker gene across muscle tissues dropped linearly, although an unusual increase was found in the pectoral muscle of females of BYC at D1 (Fig. 2B) and the thigh muscle of females of WPR at E17 (Fig. 2C). Notably, the expression level of the Myomaker gene was nearly zero at D1 (except for BYC in the pectoral muscle) due to the decrease in myoblast fusion and the basic formation of myofibers during the late embryonic stage.
The expression of the MYOD gene in the pectoral muscle and thigh muscle of the four chicken breeds was clearly lower during the embryonic stage compared with D1 (Fig. 2E-H). The overall expression trends of the MYOD gene in the males were similar to those in the females. In brief, the gene expression level increased from E11 to E15, decreased slightly from E15 to E17, and then increased sharply from E17 to D1. Unlike that of the previous two genes, the expression level of MSTN in all breeds increased greatly with embryonic development and peaked at E17. After the chickens hatched, the gene expression level of MSTN significantly declined (Fig. 2I-L), especially in CN, in which it decreased to nearly zero for both males and females.
Meat-type chicken breed had the highest gene expression of Myomaker at the crucial period
The fusion of myoblasts increased from E13 to E15 due to the highest gene expression level of Myomaker. This result was further corroborated by analyzing the myofiber morphology of chicken embryos. Histological sections of the myofibers at each time point were generated, and it could be seen that the myoblasts at E13 to E15 were in the process of rapid fusion, which was consistent with the gene expression characteristics of Myomaker. The contours of myofibers were not clear until E17 (Fig. 3), indicating that the formation of myofibers had been completed at E17, after which myofiber hypertrophy was observed.
Since the crucial period of myoblast fusion was E13 to E15, we further analyzed the differential gene expression of Myomaker among various chicken breeds. WL and BYC are the egg-type chicken and native breeds, respectively. CN and WPR are the paternal and maternal lines of modern commercial broilers, respectively. There was no obvious difference in Myomaker gene expression among the males of the four chicken breeds at E13 (Fig. 4A-B). However, the muscles of CN females had higher Myomaker gene expression than did those of the other three breeds at E13 (Fig. 4C-D). The relative expression of Myomaker in the pectoral muscle in CN males at E15 was 1.81 ± 0.24, which was extremely significantly higher than that in the WL, BYC and WPR males (P < 0.01, Fig. 4E). Similar results were found in the thigh muscle of males (Fig. 4F) and both muscle tissues of the females (Fig. 4G-H). These results indicate that the Myomaker gene expression in CN was significantly higher than that in the other three chicken breeds at the crucial period and that the data were robust.
The various gene expression of MYOD among the four chicken breeds at D1
As noted above, the expression of the MYOD gene in muscles at D1 was higher than that during the embryonic period, which showed little change in MYOD gene expression. This result suggested that the MYOD gene may more strongly regulate myogenesis after hatching. Therefore, the important time point of D1 was selected to investigate the differential gene expression of MYOD among various chicken breeds. There was an extremely significant difference in MYOD gene expression in the pectoral muscle (72.68 ± 10.34) and thigh muscle (75.20 ± 10.04) of males between WPR and the other three chicken breeds at D1 (P < 0.01, Fig. 5A-B). In addition, MYOD gene expression in the muscles of males of CN was significantly lower than that in WPR at D1 (P < 0.01, Fig. 5A-B). The pectoral muscle and thigh muscle of females of WPR still had the highest MYOD gene expression among the four chicken breeds at D1 (Fig. 5C-D). The difference in gene expression between CN females and the other chicken breed was changed compared to that of CN males at D1. In particular, the MYOD gene expression in the thigh muscle of CN females was significantly higher than that in egg-type chickens and Chinese native breeds (P < 0.01, Fig. 5D), which implied that the gene expression patterns of MYOD were slightly different between males and females at D1.
The inhibition of MSTN for the myoblast fusion
The expression level of the MSTN gene was low during the critical period of myoblast fusion, while it reached the highest level at E17, at which time the myofiber development of chicken embryos was repressed. The gene expression of MSTN in pectoral muscle of BYC males (23.10 ± 0.95) was significantly higher than that of WL and WPR males at E17 (P < 0.01, Fig. 6A). Similar results were obtained in the thigh muscle of BYC males (Fig. 6B). Interestingly, there was no significant difference in MSTN in the muscles of females among various chicken breeds at E17 (Fig. 6C-D), which indicated that MSTN gene expression in muscles might be different between male and female chicken embryos.