In search of the molecular mechanisms of myostatin
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Myostatin is a negative regulator of skeletal muscle growth. Knock-out mice have been shown to have a 200% increase in skeletal muscle mass primarily to myofiber hyperplasia and to a lesser extent, hypertrophy. Numerous mutations within myostatin have been identified which are responsible for the double-muscled phenotype in cattle. However, the molecular mechanisms whereby myostatin inhibits skeletal muscle mass are not fully understood. The goal of this research was to identify genes that are differentially expressed in double-muscled versus normal-muscled embryos in order to identify potential candidate genes that may be regulated by myostatin activated pathways. Suppressive subtractive hybridization was performed in order to identify genes that had increased expression in either double-muscled or normal-muscled bovine embryos. Crossbred Belgian Blue dams and sires were selected to minimize genetic variation at loci other than myostatin. Embryos were collected at 31 to 33 days of gestation, which is 2 to 4 days after the initial expression of myostatin. Thirty-one clones were identified that were potentially differentially expressed. Macroarray analysis of the original RNA samples confirmed that 20 of these clones were differentially expressed in the original cDNA libraries. Several of these genes have biological functions that correspond very well with myostatin's role in skeletal muscle development. However, whole embryos were utilized for the suppressive subtractive hybridization. In order to determine if they were expressed in skeletal muscle, expression levels in myoblasts and myotubes were analyzed. Only a few of the genes were expressed in myoblasts or myotubes. The genes that were not expressed may have been important during specification of the somite, or myostatin may play a systemic role and the genes are actually expressed elsewhere in the embryo. This research added potential insight about the molecular mechanisms whereby myostatin inhibits skeletal muscle mass. Furthermore, several of these genes map to quantitative trait loci known to interact with the presence or absence of myostatin. Physical mapping was completed on genes that were in QTL regions and single nucleotide polymorphisms were identified for genes in these regions. These will be used to discover if these genes contain myostatin interacting alleles.