Gene expression of Hanwoo satellite cell differentiation in longissimus dorsi and semimembranosus

Research article Open Access Gene expression of Hanwoo satellite cell differentiation in longissimus dorsi and semimembranosus Sara de las Heras-Saldana†1, Ki Yong Chung†2, Seung Hwan Lee3Email author and Cedric Gondro4Email authorView ORCID ID profile †Contributed equally BMC Genomics201920:156 https://doi.org/10.1186/s12864-019-5530-7 ©  The Author(s). 2019 Received: 3 July 2018Accepted: 13 February 2019Published: 26 February 2019 Abstract Background Korean Hanwoo cattle are known for their high meat quality, especially their high intramuscular fat compared to most other cattle breeds. Different muscles have very different meat quality traits and a study of the myogenic process in satellite cells can help us better understand the genes and pathways that regulate this process and how muscles differentiate. Results Cell cultures of Longissimus dorsi muscle differentiated from myoblast into multinucleated myotubes faster than semimembranosus. Time-series RNA-seq identified a total of 13 differentially expressed genes between the two muscles during their development. These genes seem to be involved in determining muscle lineage development and appear to modulate the expression of myogenic regulatory factors (mainly MYOD and MYF5) during differentiation of satellite cells into multinucleate myotubes. Gene ontology enriched terms were consistent with the morphological changes observed in the histology. Most of the over-represented terms and genes expressed during myoblast differentiation were similar regardless of muscle type which indicates a highly conserved myogenic process albeit the rates of differentiation being different. There were more differences in the enriched GO terms during the end of proliferation compared to myoblast differentiation. Conclusions The use of satellite cells from newborn Hanwoo calves appears to be a good model to study embryonic myogenesis in muscle. Our findings provide evidence that the differential expression of HOXB2, HOXB4, HOXB9, HOXC8, FOXD1, IGFN1, ZIC2, ZIC4, HOXA11, HOXC11, PITX1, SIM2 and TBX4 genes could be involved in the differentiation of Longissimus dorsi and Semimembranosus muscles. These genes seem to modulate the muscle fate of the satellite cells during myogenesis through a differential expression profile that also controls the expression of some myogenic regulatory factors (MYOD and MYF5). The number of differentially expressed genes across time was unsurprisingly large. In relation to the baseline day 0, there were 631, 155, 175, 519 and 586 DE genes in LD, while in SM we found 204, 0, 615, 761 and 1154 DE genes at days 1, 2, 4, 7 and 14 respectively. Keywords HanwooSatellite cellMuscle differentiationMyogenesisRNA-seq Background The Korean Hanwoo cattle is known for its meat quality and high marbling ability (intramuscular fat) [1]. Meat quality (e.g. juiciness, tenderness, flavor) is mainly determined by the structure of the meat and its fatty acid composition, both of which vary widely across muscle groups [1–3]. Transcriptional analysis has been very useful to characterize gene expression differences in muscles from different breeds, with divergent phenotypes and across muscle groups [4]. It has also helped us understand the genetic mechanisms that underpin muscle development (myogenesis) [5] and how muscle developmental differences observed at the proteomic [6] and transcriptomic levels [7, 8] can affect production traits. Myogenesis is mainly controlled by the Myogenic Regulatory Factors (MRFs) that modulate myoblast proliferation, migration and fusion [9]. There are four MRFs (MYF5, MYOD, MRF4/MYF6, and MYOG), however there are also several other genes that contribute to the regulation of growth and differentiation [10] and there are still many unknowns surrounding the exact molecular mechanisms involved in muscle differentiation – particularly which genes change expression and when do these changes occur – that ultimately lead to the morphological and phenotypic differences observed across the different types of muscle. Satellite cells are myogenic stem cells with the potential to self-renew and produce differentiated progeny; for this reason, these cells play an essential role in postnatal growth, muscle regeneration and hypertrophy. Myogenesis of satellite cells is a good model to study changes in gene expression over time and how they relate to muscle proliferation and differentiation [7, 8, 11, 12]. The combination of RNA sequencing with histological techniques allows for a deeper understanding of the mechanisms mediating differentiation of satellite cells into different muscle types revealing their gene expression profile and regulatory mechanisms at specific differentiation stages. A better understanding of these mechanisms is important for developmental biology and can assist in the development of therapeutic protocols in muscle. In this study, muscle biopsies were performed on three Hanwoo calves to extract muscle satellite cells from Longissimus dorsi (LD) and Semimembranosus (SM). Cells from these two muscles were cultured and allowed to differentiate into myotubes. This process was studied using RNA-seq and morphological measurements across six time points. The main objective of this study was to describe the expression profile of genes during early muscle differentiation in Hanwoo and to find differentially expressed genes between LD and SM muscles that may be involved in modulating muscle fate. We found that the gene expression profile over time is similar in both muscles which indicates a highly conserved myogenic process. However, our results indicate that the two muscles differentiate at different rates and that 13 genes seem to be involved in determining the fate of the satellite cells into one muscle type or another. Identification of the biological triggers in the early stages of muscle development can be of value to understand the different characteristics of muscles in adult cattle. Results Morphological analysis The in vitro bovine muscle satellite cells (MSC) proliferated until they reached 60–70% confluence after four or 5 days of culture. The MSC were then treated with the differentiation medium and this timepoint was taken as day 0 (Fig. 1). Differentiation of bovine myoblasts began between 2 and 4 days later. LD formed multinucleated myotubes with significantly higher differentiation indexes compared to SM at days 3, 4 and 7 (Fig. 2) which suggests a faster differentiation process in LD myoblasts. This faster differentiation in LD also hints at a faster proliferation rate in comparison to SM, however it was not measured in this study. On day 7, the myotubes of both muscles went through significant morphological changes by fusing to form mature multinucleated myotubes. There was also a significant reduction in the area occupied by the myotubes on day 7 in comparison to day 4 (Fig. 2). The differentiation indexes were calculated just for d (...truncated)