Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity

Abstract

Background: Postnatal muscle growth is largely depending on the number and size of muscle fibers. The number of myofibers and to a large extent their metabolic and contractile properties, which also influence their size, are determined prenatally during the process of myogenesis. Hence identification of genes and their networks governing prenatal development of skeletal muscles will provide insight into the control of muscle growth and facilitate finding the source of its variation. So far most of the genes involved in myogenesis were identified by in vitro studies using gene targeting and transgenesis. Profiling of transcriptome changes during the myogenesis in vivo promises to obtain a more complete picture. In order to address this, we performed transcriptome profiling of prenatal skeletal muscle using differential display RT-PCR as on open system with the potential to detect novel transcripts. Seven key stages of myogenesis (days 14, 21, 35, 49, 63, 77 and 91 post conception) were studied in two breeds, Pietrain and Duroc, differing markedly in muscularity and muscle structure.

Results: Eighty prominent cDNA fragments were sequenced, 43 showing stage-associated and 37 showing breed-associated differences in the expression, respectively. Out of the resulting 85 unique expressed sequence tags, EST, 52 could be assigned to known genes. The most frequent functional categories represented genes encoding myofibrillar proteins (8), genes involved in cell adhesion, cell-cell signaling and extracellular matrix synthesis/remodeling (8), genes regulating gene expression (8), and metabolism genes (8). Some of the EST that showed no identity to any known transcripts in the databases are located in introns of known genes and most likely represent novel exons (e.g. HMGA2). Expression of thirteen transcripts along with five reference genes was further analyzed by means of real-time quantitative PCR. Nine of the target transcripts showed higher than twofold differences in the expression between the two breeds (GATA3, HMGA2, NRAP, SMC6L1, SPP1, RAB6IP2, TJP1 and two EST).

Conclusion: The present study revealed several genes and novel transcripts not previously associated with myogenesis and expands our knowledge of genetic factors operating during myogenesis. Genes that exhibited differences between the divergent breeds represent candidate genes for muscle growth and structure.

Figure 1

Expression of reference genes (A-E) and genes showing stage-associated differential display RT-PCR patterns (F-H) obtained by qPCR. Histograms show normalized relative gene expression in Pietrain and Duroc embryos/Musculus longissimus dorsi at seven prenatal stages and in the adults (primary y-axis). For some genes data on expression in adults is missing. Open boxes and diamonds show absolute values of the Pietrain vs. Duroc expression ratios at each stage (log2(Pi/Du)). Open diamonds indicate higher expression in Duroc compared to Pietrain individuals and open boxes indicate the reverse situation (secondary y-axis).

Figure 2

Expression of ten genes showing breed-associated differences in differential display RT-PCR patterns obtained by qPCR. Histograms show normalized relative gene expression in Pietrain and Duroc embryos/Musculus longissimus dorsi at seven prenatal stages and in the adults (primary y-axis). For some genes data on expression in adults is missing. Open boxes and diamonds show absolute values of the Pietrain vs. Duroc expression ratios at each stage (log2(Pi/Du)). Open diamonds indicate higher expression in Duroc compared to Pietrain individuals and open boxes indicate the reverse situation (secondary y-axis).