ABSTRACT: Integrating transcriptome and proteome profiles of prenatal muscle tissue to reveal the postnatal growth differences in pigsof Pig Muscle Tissue
Project description:To obtain an overview of the methylome landscape in the developing pig skeletal muscle, 81 high-quality whole-genome bisulfite sequencing(WGBS) libraries that covered 27 developmental stages (3 biological replicates per stage) from embryonic day 33 (E33) to postnatal day 180 (D180) were produced by whole-genome bisulfite sequencing.
Project description:Background Neurofibromatosis type 1 (NF1) is a multi-organ disease caused by mutations in Neurofibromin (NF1). Amongst other features, NF1 patients frequently show reduced muscle mass and strength, impairing patients’ mobility and increasing the risk of fall. The role of Nf1 in muscle and the cause for the NF1-associated myopathy is mostly unknown. Methods To dissect the function of Nf1 in muscle, we created muscle-specific knockout mouse models for Nf1, inactivating Nf1 in the prenatal myogenic lineage either under the Lbx1 promoter or under the Myf5 promoter. Mice were analyzed during pre-and postnatal myogenesis and muscle growth. Results Nf1Lbx1 and Nf1Myf5 animals showed only mild defects in prenatal myogenesis. Nf1Lbx1 animals were perinatally lethal, while Nf1Myf5 animals survived up to approx. 25 weeks. Nf1Myf5 animals showed decreased postnatal growth, reduced muscle size, and fast fiber atrophy. Proteome and transcriptome analysis of muscle tissue indicated decreased protein synthesis and increased proteasomal degradation, and decreased glycolytic and increased oxidative activity in muscle tissue. Real-time respirometry demonstrated enhanced oxidative metabolism in Nf1Myf5 muscles concomitant to a fiber type shift from type 2B to type 2A and type 1. Nf1Myf5 muscles showed hallmarks of mild oxidative stress and increased activation of AMPK indicating an energy deficit, increased expression of atrogenes and decreased activation of mTORC1. Proteome and transcriptome analysis indicated that oxidative fibers mainly relied on fatty acid catabolism. Inline, Nf1Myf5 animals showed a drastic reduction of white, but not brown, adipose tissue. Conclusions Our results demonstrate a cell-autonomous role for Nf1 in myogenic cells during postnatal muscle growth required for metabolic and proteostatic homeostasis. Furthermore, Nf1 deficiency in muscle leads to cross-tissue communication and mobilization of lipid reserves.
Project description:Purpose: MicroRNAs (miRNAs) are non-coding small miRNA ~22 nucleotides in length and play a vital role in muscle development by binding to messenger RNAs (mRNAs). Large White (LW, a lean type pig) and Meishan pigs (MS, a Chinese indigenous obese breed) have significant postnatal phenotype differences in growth rate, muscle mass and meat quality, and theses difference are programmed during prenatal muscle development.To shed directly light on the miRNA transcriptome difference in prenatal muscles between these two distinct pig breeds. Methods: skeletal musle miRNA profiles of Meishan pig and large white pig were generated by deep sequencing, in triplicate, using Illumina Hiseq2000 . The sequence reads that passed quality filters were analyzed at the mature miRNA level . qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: We measure the myofiber phenotypes of LW and MS at developmental stages of 35, 55 and 90 days post-conception (dpc). Results shows that the myogenesis process is more intense in MS than in LW at 35 dpc. To investigate the role of miRNAs involved in regulating muscle development at earlier stages of myogenesis and decipher the miRNAs transcriptome difference between LW and MS, here, the miRNAomes of longissimus dorsi muscle collected at 35 dpc from female LW and MS were analyzed by deep sequencing. Overall, we identified 1147 unique miRNAs comprising 434 known miRNAs, 239 conserved miRNAs and 474 candidate miRNAs. Expression analysis of the 10 most abundant miRANs in every library indicated that functional miRNAome may be smaller and tend to be highly expressed. These set of miRNA may play house keeping roles that were involved in myogenesis. A total of 87 miRNAs were significantly differentially expressed between LW and MS (reads > 1000, P < 0.05). Gene ontology (GO) and KEGG pathway enrichment analysis revealed that the differentially expressed miRNAs (DE miRNAs) were associated mainly with muscle contraction, WNT, mTOR, and MAPK signaling pathways. In addition, the expression patterns of myomiRs (miR-1,-133,-206) at three prenatal stages (35,55 and 90 dpc) were determined using qRT-PCR, Notably, ssc-miR-133 was significantly more highly expressed in LW pig skeletal muscle at all prenatal stages compared with its expression in LW pig skeletal muscle. We concluded that the higher expression levels of miR-133 in MS, possibly regulated by myoD, are potential strategies for improvement of myogenesis in earlier prenatal stages. Conclusions: our results add new information to existing data on porcine miRNAs and be helpful to investigate the dominant (main functional) muscle-related miRNAs sets in different pig breeds. In addition, this study provide a profound knowledge on the role of miRNAs in prenatal muscle development, which could provide great reference value for pork quality improvement.
Project description:To obtain an overview of the transcriptome landscape in developing pig skeletal muscle, 81 high-quality transcriptome libraries that covered 27 developmental stages (3 biological replicates per stage) in pig skeletal muscle were produced by strand-specific rRNA-depleted total RNA sequencing (RNA-seq). We generated 8.59 billion paired-end reads (150 bp × 2) covering 1.24 Tb of sequence for RNA-seq.
Project description:Obese and lean pig show breed-specific traits in muscle growth and meat quality. However, the mechanisms underlying remains unclear. Here, we reported the first genome-wide muscle development research from embryonic to 6 months old between Lantang (obese) and Landrace (lean) using Solexa/Illumina’s Genome Analyzer. We obtained 4.22±0.9×107 total reads per sample with approximately 5×106 distinct tags. Filtered adaptor tags, empty reads, low quality tags and tags of copy number =1, we obtained 3.84±0.9×107 clean total tags with 1.5±0.5×106 clean distinct tags. 20 libraries of longissimus muscle samples were sequenced in Lantang and Landrace at days 35, 49, 63, 77, 91 prenatal and 2, 28, 90, 120, 180 postnatal days.
Project description:Obese and lean pig breeds showed obvious phenotypic variations and physiological differences in skeletal muscle growth. Prenatal muscle development programs postnatal performance. In this study we initially conducted full transcriptional profiling of prenatal skeletal muscle from Tongcheng pigs (obese) and Landrace pigs (lean) at 33, 65 and 90 days post coitus (dpc), using long serial analysis of gene expression (LongSAGE). We subsequently sequenced 317,115 LongSAGE tags and identified 1400 and 1201 differentially expressed transcripts that showed eight expression patterns for Tongcheng and Landrace, respectively. These two breeds had more significant differences in their gene expression profiles at 65 than that at 33 and 90 dpc. We also identified 532, 653 and 459 transcripts that were differentially expressed at 33, 65 and 90 dpc between the two breeds, respectively. The cellular function of the differentially expressed transcripts that matched annotated genes revealed that each stage had a uniquely altered profile between the two breeds in various functional categories including muscle fiber constitute and contraction, apoptosis, protein synthesis, and signalling transduction. Our results suggest that skeletal muscle development potentially has a greater lag in its growth rate at 33-65 dpc in Tongcheng pigs when compared with their Landrace counterparts. Our analyses therefore not only will provide valuable resources in the further identification of candidate genes for meat production traits, but also assist in the elucidation of the development of prenatal skeletal muscle in pigs and other vertebrates. Keywords: comparative transcriptome analysis