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• myod and myf5 were predominant in skeletal muscle with the highest expression in white muscle. • myod and myf5 mRNAs were the highest at 10 dph and the segmentation period, respectively. • LPS affected the expression of muscle related genes, and activated satellite cells. Myod and Myf5 are muscle-specific basic helix-loop-helix (bHLH) transcription factors that play essential roles in regulating skeletal muscle development and growth. In order to investigate potential function of myod and myf5 of Megalobrama amblycephala , an economically important freshwater fish species, in the present study, we characterized the sequences and expression profiles of M. amblycephala myod and myf5. The open reading frame (ORF) sequences of myod and myf5 encoded 275 and 240 amino acids, respectively, possessing analogous structure with the highly conserved domains, bHLH and C-terminal helix III domains. Spatio-temporal expression patterns revealed that myod and myf5 were predominant in skeletal muscle with the highest expression in white muscle, and the highest at 10 days post-hatching (dph) and the segmentation period, respectively. Furthermore, we evaluated the effects of lipopolysaccharide (LPS) on the expression of muscle-related genes in white and red muscle, and proliferation and differentiation of satellite cells. The myod , myf5 and pax-7 expression generally increased and then decreased with increase of LPS concentration and treatment time in red muscle, while these genes showed inconsistent expression patterns in white muscle. In addition, LPS administration caused the frequency increase of satellite cells in red and white muscle especially at 3 and 7 days after LPS-injection. [ABSTRACT FROM AUTHOR]

Abstract HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream (Megalobrama amblycephala) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation. Highlights • Megalobrama amblycephala phd1 was not induced by hypoxia in most tissues. • Two Phd1 isoforms generated by alternative initiation were identified in the nucleus. • Only the senior isoform promoted cell proliferation. [ABSTRACT FROM AUTHOR]

The economic and biological significance of blunt snout bream ( Megalobrama amblycephala ) makes this species important to explore the underlying molecular mechanism of hypoxia response. In the present study, we compared the transcriptional responses to serious hypoxia in skeletal muscle among hypoxia tolerant (MT), sensitive (MS) and control (without hypoxia treatment, MC) M. amblycephala obtained according to the time difference of losing balance after hypoxia treatment. A total of 88,200,889 clean reads were generated and assembled into 44,493 unigenes. Transcriptomic comparison revealed 463 genes differentially expressed among different groups. A similar hypoxia-induced transcription patterns suggested a common hypoxia response involved in cell cycle, p53 signaling pathway, apoptosis, heart contraction and blood circulation. Interesting, four genes, heat shock protein beta-8 ( hspb8 ), cysteine/serine-rich nuclear protein 1 ( csrnp1 ), salt-inducible kinase 1 ( sik1 ), and visinin-like 1a ( vsnl1a ) were up-regulated in MT Vs MC but down-regulated in MS Vs MC. Additionally, FoxO signaling pathway was significantly enriched only in MT Vs MC. These results not only provided the first insights into the mechanism that muscle tissue coped with the hypoxia stress in cyprinid species, but offered a theory base for breeding of M. amblycephala with hypoxia-resistant traits. [ABSTRACT FROM AUTHOR]

PHD3 is a hydroxylase that hydroxylates prolyl residues on hypoxia-inducible factors (HIFs) in mammals. In this study, the full-length cDNA and promoter sequences of Megalobrama amblycephala PHD3 gene were isolated by a modified RACE method. PHD3 cDNA was 1622 bp in length, including an ORF of 717 bp encoding 238 amino acid residues. The semi-quantitative PCR results suggested that PHD3 was highly expressed in liver in the normal condition, while after hypoxia treatment this gene was significantly increased in all analyzed tissues. PHD3 was detected only in the initial stages of M. amblycephala embryo development. In addition, the presence of another alternatively processed PHD3 transcript, designated PHD3Δ1 was observed in the process of analyzing the expression of PHD3 . Both PHD3 and PHD3Δ1 were up-regulated under hypoxia, and had five the hypoxia response elements (HREs) by in silico scanning on the promoter. Further luciferase assay indicated that all HREs significantly responded to hypoxia. Taken together, these results suggest that PHD3 plays important roles in hypoxia response and early embryo development of M. amblycephala . [ABSTRACT FROM AUTHOR]

• Muscle injury promoted proliferation of Pax7+ cells in M. amblycephala. • Total 6729 DEGs were identified by RNA-seq after muscle injury. • Cytoskeleton remodeling and lipid metabolism were found to be important for muscle repair after injury. Skeletal muscle myogenesis and injury-induced muscle regeneration contribute to muscle formation. Skeletal muscle stem cells, termed satellite cells (SCs), proliferate to repair injured muscle. To identify the molecular mechanism of regeneration after muscle injury as well as the genes related to muscle development in fish, in this study, the immunohistochemistry and the high-throughput RNA sequencing (RNA-seq) analysis were performed after Megalobrama amblycephala muscle was injured by needle stab. The results showed that paired box7-positive (Pax7+) SCs increased, and peaked at 96 to 144 h-post injury (hpi). The 6729 differentially expressed genes (DEGs), including 2125 up-regulated and 4604 down-regulated genes were found. GO terms significantly enriched by DEGs contained intercellular connections, signaling transduction and enzyme activity. KEGG enrichment analysis showed that most of the pathways were related to immunity, metabolism and cells related molecules, including actin skeleton regulation, Epstein Barr virus infection and plaque adhesion. The WGCNA results revealed that actin cytoskeleton and lipid metabolism related genes probably played crucial roles during repair after muscle injury. Collectively, all these results will provide new insights into the molecular mechanisms underlying muscle injury repair of fish. [ABSTRACT FROM AUTHOR]

Abstract: Cardiac troponin T (TNNT2), as a member of troponin superfamily, plays important roles during early cardiogenesis, and contraction and relaxation of myocardial cells. In this study, two alternatively spliced variants of Megalobrama amblycephala TNNT2 were identified showing a difference of 19 amino acids in the N-terminal hypervariable region. The longer cDNA (TNNT2-1) was 1118bp, encoding 284 amino acid residues, contained conserved central tropomyosin-binding region, cardiac specific signal and C-terminal segments except the N-terminal hypervariable region. The TNNT2 transcripts first appeared at 16hours post-fertilization (hpf) peaking at 28hpf (onset of heartbeat). In addition, strong expression of TNNT2 was found in the cardiac muscle. After nitrite exposure, the increased TNNT2 expression levels in the heart indicated that nitrite might induce cardiac injury. Results of semi-quantitative RT-PCR indicated that the two alternatively spliced variants existed in early development stages since their first appearance at 16hpf and heart, spleen, headkiney of M. amblycephala. The shorter transcript (TNNT2-2) was proved to be dominant in the embryos and heart of M. amblycephala, furthermore, the increase of TNNT2 expression level in the heart after nitrite exposure was mainly caused by TNNT2-2. [Copyright &y& Elsevier]

Abstract: Chemokine (C–X–C motif), receptor 4 (CXCR4), a member of the family of seven transmembrane G-protein-coupled receptors, plays important roles in immunomodulation, organogenesis, hematopoiesis, and derailed cerebellar neuron migration. We characterized the sequences and expression profiles of CXCR4b in Megalobrama amblycephala. The full-length cDNA was 1638bp, encoding 353 amino acid residues. Multiple alignment and phylogenetic analysis indicated that M. amblycephala CXCR4b contained the similar conservative sequences and motifs with other organisms. The CXCR4b expression in different development stages of M. amblycephala showed the mRNA levels before hatching and at 62h post fertilization (hpf) were significantly higher than at other post hatching stages (P <0.05). Besides, CXCR4b was constitutively expressed in a wide range of tissues, at higher levels in headkidney, liver, intestine spleen, blood and gill, where a larger number of immune cells including lymphocytes and macrophages reside, suggesting its specific roles in inflammatory responses. The CXCR4b expression after high nitrite concentration (ρNO2−-N: 20.29mg/L) exposure supported a potential pro-inflammatory function for CXCR4b. In order to identify the better population with immune property for breeding, we compared the tissue expression of CXCR4b among Liangzi Lake population (L), Yuni Lake population (Y) and Poyang Lake population (P), it was indicated that the expression levels in the population Y were obviously higher than that of the other two populations. [Copyright &y& Elsevier]