Gene co-expression network analysis of myostatin regulation at three different mouse developmental timepoints

Myostatin (Mstn) knockout mice exhibit large increases in skeletal muscle mass. However, relatively few of the genes that mediate or modify MSTN effects are known. In this study, we performed co-expression network analysis using whole transcriptome microarray data from Mstn-null and wild-type mice to identify genes involved in important biological processes and pathways related to skeletal muscle and adipose development.Genes differentially expressed between wild-type and Mstn-null mice were identified at 13.5 d.p.c. and 35 days after birth (d35) and further analyzed for shared DNA motifs using DREME. Known and novel transcription factor binding sites involved in MSTN signal transduction were enriched in differentially expressed genes. The use of regulatory impact factor (RIF), phenotypic impact factor (PIF), and differential hubbing analyses with the partical correlation and information theory (PCIT) co-expression analysis methods identified both known and potentially novel regulators of muscle development. These results provide new details of how MSTN may mediate transcriptional regulation as well as insight into novel regulators of MSTN signal transduction that merit further study regarding their physiological roles in muscle and adipose development. Mstn-null and wild-type mice were reared, sacrificed by CO2 asphyxiation (embryonic timepoints) or cervical dislocation (d35), and tissues were collected from whole embryos (13.5 d.p.c.), hindquarters (17.5 d.p.c.), or pectoralis muscle (d35). After collection, muscles were flash-frozen in liquid nitrogen as previously described in Steelman et al., 2006. FASEB J. Mar;20(3):580-2. For microarray analysis, 10 μg of RNA from each of three individual animals was pooled to give five pools (N=15, total number of arrays=5) for each genotype/timepoint combination.