Expressed Sequence Tags for Bovine Muscle Satellite Cells, Myotube Formed-Cells and Adipocyte-Like Cells

<div><p>Background</p><p>Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.</p> <p>Results</p><p>A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of <i>dimethylarginine</i><i>dimethylaminohydrolase2</i> (<i>DDAH2</i>) during myogenesis and <i>hemoglobin</i><i>subunit</i><i>alpha2</i> (<i>HBA2</i>) during transdifferentiation in C2C12 were assayed as a case study. <i>DDAH2</i> was up-regulated during myognesis and knockdown of <i>DDAH2</i> by siRNA significantly decreased myogenin (<i>MYOG</i>) expression corresponding with the slight change in cell morphology. In contrast, <i>HBA2</i> was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and <i>CD36</i> mRNA expression upon knockdown assay.</p> <p>Conclusion</p><p>In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.</p> </div

KOG analysis of ESTs in MSC, MFC and ALC.

<div><p>The functions of genes were categorized and each function is represented by the symbols given below: .</p> <p>[J] Translation, ribosomal structure and biogenesis, [A] RNA processing and modification, [L] replication, recombination and repair, [B] chromatin structure and dynamics, [Y] nuclear structure, [V] defense mechanisms, [T] signal transduction mechanisms, [M] cell wall/membrane/envelope biogenesis, [N] cell motility, [Z] cytoskeleton, [W] extracellular structures, [U] intracellular trafficking, secretion, and vesicular transport, [O] posttranslational modification, protein turnover, chaperones, [C] energy production and conversion, [G] carbohydrate transport and metabolism, [E] amino acid transport and metabolism, [F] nucleotide transport and metabolism, [H] coenzyme transport and metabolism, [I] lipid transport and metabolism, [P] inorganic ion transport and metabolism, [R] general function prediction only, [S] function unknown. The X-axis represents percentage and Y axis represents functional category. </p></div

<i>DDAH2</i> knockdown in C2C12 cells during myogenesis.

<p>mRNA expression of <i>DDAH2</i> and <i>MYOG</i> after <i>DDAH2</i>_kd during differentiation in C2C12 at Day 2 (A). Representative cell picture showing morphological changes in <i>DDAH2</i>_kd cells (B). Mock represents control (mean ± S.D., n= 3). <i>p</i>-value indicates the statistical significance of the data and different letters show significant differences among groups.</p

<i>HBA2</i> knockdown in C2C12 cells during transdifferentiation.

<p>(A) mRNA expression of <i>HBA2</i> after <i>HBA2</i>_kd during transdifferentiation in C2C12 at Day 1, and (B) <i>HBA2</i>_kd effect on mRNA expression of different adipogenic marker genes.(C) Cell picture following intracellular lipid staining by O-R-O on Day 5 during transdifferentiation in HBA2_kd and Mock cells. Quantification of O-R-O at 510 nm. Control represents Mock (mean ± S.D., n= 3). <i>p</i>-value indicates the statistical significance of the data and different letters show significant differences among groups.</p

Schematic diagram for EST data analysis.

<p>Schematic diagram for EST data analysis.</p

mRNA expression of genes identified with higher EST numbers.

<p>C2C12 cells treated with differentiation and transdifferentiation media at 80% confluence were harvested at different time points. Real time PCR was carried out with cDNA synthesized from1µg of total RNA. mRNA expression analysis of most of the genes showed more than 2 fold induction during MFCs (A) and ALCs (B) formation, respectively. Western blot analysis of <i>DDAH2</i> shows a gradual increase in protein expression with time (C). Cellular localization of DDAH2 by immunocytochemistry during myogenesis. The first column shows cell pictures at Day 0 and Day 3. The second column shows expression of <i>DDAH2</i> and the third column shows a merged image of DAPI-stained nuclei and <i>DDAH2</i> IF (D).Western blot analysis of <i>HBA2</i> expression during transdifferentiation confirms its protein level expression (E). Cytoplasmic localization of HBA2 by immunocytochemistry during transdifferentiation. The first column shows cell pictures at Day 0 and Day 2, the second column shows expression of <i>HBA2</i> IF and the third column shows a merged image of DAPI-stained nuclei and <i>HBA2</i> IF (F). DDAH2 and HBA2 immunohistochemistry of bovine skeletal muscle (G and H). Day 0 represents control (mean ± S.D., n= 3). <i>p</i>-value indicates the statistical significance of the data and different letters show significant differences among groups.</p