Identification of the crucial molecular events during the large-scale myoblast fusion in sheep

2014 ◽  
Vol 46 (12) ◽  
pp. 429-440 ◽  
Author(s):  
Caihong Wei ◽  
Li Li ◽  
Hongwei Su ◽  
Lingyang Xu ◽  
Jian Lu ◽  
...  

It is well known that in sheep most myofibers are formed before birth; however, the crucial myogenic stage and the cellular and molecular mechanisms underpinning phenotypic variation of fetal muscle development remain to be ascertained. We used histological, microarray, and quantitative real-time PCR (qPCR) methods to examine the developmental characteristics of fetal muscle at 70, 85, 100, 120, and 135 days of gestation in sheep. We show that day 100 is an important checkpoint for change in muscle transcriptome and histomorphology in fetal sheep and that the period of 85–100 days is the vital developmental stage for large-scale myoblast fusion. Furthermore, we identified the cis-regulatory motifs for E2F1 or MEF2A in a list of decreasingly or increasingly expressed genes between 85 and 100 days, respectively. Further analysis demonstrated that the mRNA and phosphorylated protein levels of E2F1 and MEF2A significantly declined with myogenic progression in vivo and in vitro. qRT-PCR analysis indicated that PI3K and FST, as targets of E2F1, may be involved in myoblast differentiation and fusion and that downregulation of MEF2A contributes to transition of myofiber types by differential regulation of the target genes involved at the stage of 85–100 days. We clarify for the first time the timing of myofiber proliferation and development during gestation in sheep, which would be beneficial to meat sheep production. Our findings present a repertoire of gene expression in muscle during large-scale myoblast fusion at transcriptome-wide level, which contributes to elucidate the regulatory network of myogenic differentiation.

2002 ◽  
Vol 159 (1) ◽  
pp. 113-122 ◽  
Author(s):  
Bernd Martin ◽  
Richard Schneider ◽  
Stefanie Janetzky ◽  
Zoe Waibler ◽  
Petra Pandur ◽  
...  

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.


2018 ◽  
Vol 115 (42) ◽  
pp. E9802-E9811 ◽  
Author(s):  
Jian Jun Jin ◽  
Wei Lv ◽  
Pan Xia ◽  
Zai Yan Xu ◽  
An Dai Zheng ◽  
...  

Although many long noncoding RNAs (lncRNAs) have been identified in muscle, their physiological function and regulatory mechanisms remain largely unexplored. In this study, we systematically characterized the expression profiles of lncRNAs during C2C12 myoblast differentiation and identified an intronic lncRNA,SYISL(SYNPO2intron sense-overlapping lncRNA), that is highly expressed in muscle. Functionally,SYISLpromotes myoblast proliferation and fusion but inhibits myogenic differentiation.SYISLknockout in mice results in significantly increased muscle fiber density and muscle mass. Mechanistically,SYISLrecruits the enhancer of zeste homolog 2 (EZH2) protein, the core component of polycomb repressive complex 2 (PRC2), to the promoters of the cell-cycle inhibitor genep21and muscle-specific genes such as myogenin (MyoG), muscle creatine kinase (MCK), and myosin heavy chain 4 (Myh4), leading to H3K27 trimethylation and epigenetic silencing of target genes. Taken together, our results reveal thatSYISLis a repressor of muscle development and plays a vital role in PRC2-mediated myogenesis.


2010 ◽  
Vol 298 (5) ◽  
pp. C1100-C1108 ◽  
Author(s):  
Cheng Liu ◽  
Robert P. Gersch ◽  
Thomas J. Hawke ◽  
Michael Hadjiargyrou

Mustn1 (Mustang, musculoskeletal temporally activated novel gene) was originally identified in fracture callus tissue, but its greatest expression is detected in skeletal muscle. Thus, we conducted experiments to investigate the expression and function of Mustn1 during myogenesis. Temporally, quantitative real-time PCR analysis of muscle samples from embryonic day 17 to 12 mo of age reveals that Mustn1 mRNA expression is greatest at 3 mo of age and beyond, consistent with the expression pattern of Myod. In situ hybridization shows abundant Mustn1 expression in somites and developing skeletal muscles, while in adult muscle, Mustn1 is localized to some peripherally located nuclei. Using RNA interference (RNAi), we investigated the function of Mustn1 in C2C12 myoblasts. Though silencing Mustn1 mRNA had no effect on myoblast proliferation, it did significantly impair myoblast differentiation, preventing myofusion. Specifically, when placed in low-serum medium for up to 6 days, Mustn1-silenced myoblasts elongated poorly and were mononucleated. In contrast, control RNAi-treated and parental myoblasts presented as large, multinucleated myotubes. Further supporting the morphological observations, immunocytochemistry of Mustn1-silenced cells demonstrated significant reductions in myogenin (Myog) and myosin heavy chain (Myhc) expression at 4 and 6 days of differentiation as compared with control and parental cells. The decreases in Myog and Myhc protein expression in Mustn1-silenced cells were associated with robust (∼3-fold or greater) decreases in the expression of Myod and desmin ( Des), as well as the myofusion markers calpain 1 ( Capn1), caveolin 3 ( Cav3), and cadherin 15 (M-cadherin; Cadh15). Overall, we demonstrate that Mustn1 is an essential regulator of myogenic differentiation and myofusion, and our findings implicate Myod and Myog as its downstream targets.


2019 ◽  
Vol 20 (20) ◽  
pp. 5161
Author(s):  
Jianyu Ma ◽  
Caifang Ren ◽  
Hua Yang ◽  
Jie Zhao ◽  
Feng Wang ◽  
...  

The complement 1q binding protein C (C1QBP), also known as p32, is highly expressed in rapidly growing tissues and plays a crucial role in cell proliferation and apoptosis. However, there are no data interpreting its mechanisms in muscle development. To investigate the role of p32 in sheep muscle development, an 856 bp cDNA fragment of p32 containing an 837 bp coding sequence that encodes 278 amino acids was analyzed. We then revealed that the expression of p32 in the longissimus and quadricep muscles of fetal sheep was more significantly up-regulated than expression at other developmental stages. Furthermore, we found that the expression of p32 was increased during myoblasts differentiation in vitro. Additionally, the knockdown of p32 in sheep myoblasts effectively inhibited myoblast differentiation, proliferation, and promoted cell apoptosis in vitro. The interference of p32 also changed the energy metabolism from Oxidative Phosphorylation (OXPHOS) to glycolysis and activated AMP-activated protein kinase (AMPK) phosphorylation in sheep myoblasts in vitro. Taken together, our data suggest that p32 plays a vital role in the development of sheep muscle and provides a potential direction for future research on muscle development and some muscle diseases.


2021 ◽  
pp. 1-9
Author(s):  
Fengsong Liu ◽  
Xiaojian Pang ◽  
Ziqi Yu ◽  
Kai Wang

PURPOSE: To explore the exact molecular mechanisms underline osteosarcoma (OS) patients with lung metastases. METHODS: The differentially expressed gene (DEG) as well as differentially expressed miRNAs (DEMs) for OS lung metastases were deeply investigated with two independent sources of databases (GEO dataset and clinical participants); The enriched biological processes and signaling pathways were explored; the miRNAs-mRNAs network was constructed; the functions of potential DEGs and DEMs were also verified with external analysis. RESULTS: The OS patients with lung metastases displayed 323 DEGs as C-C motif chemokine ligand 3 (CCL3), sorting nexin 10 (SNX10), alpha-2-macroglobulin (A2M), carboxypeptidase E (CPE), Rap guanine nucleotide exchange factor 4 (RAPGEF4), PDZ domain containing 2 (PDZD2), calpain 10 (CAPN10), four and a half LIM domains 2 (FHL2), alkaline phosphatase, biomineralization associated (ALPL), interleukin 6 (IL6), solute carrier family 26 member 1 (SLC26A1) as well as smoothened, frizzled class receptor (SMO) were significant differentially expressed. At the same time, 21 DEMs were potential for the progress of OS lung metastasis with hsa-miR-638, hsa-miR-451, hsa-miR-486-5p, hsa-miR-134 and hsa-miR-648 were significant distinct. It could been shown that hsa-miR-638 manipulated the largest number of target genes. The functions of hsa-miR-638 and target mRNAs for the development of lung metastasis in OS could be confirmed by quantitative Real-time PCR analysis. CONCLUSION: This integrated study hypothesized several miRNA dependent signaling pathway for OS patients with lung metastases and initiated a potential strategy for better understanding the lung metastases in clinic.


2011 ◽  
Vol 301 (1) ◽  
pp. C195-C203 ◽  
Author(s):  
Craig McFarlane ◽  
Gu Zi Hui ◽  
Wong Zhi Wei Amanda ◽  
Hiu Yeung Lau ◽  
Sudarsanareddy Lokireddy ◽  
...  

Myostatin, a member of the transforming growth factor-β superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.


Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 961 ◽  
Author(s):  
Rohan Kar ◽  
Niraj Kumar Jha ◽  
Saurabh Kumar Jha ◽  
Ankur Sharma ◽  
Sunny Dholpuria ◽  
...  

Notch signaling is a primitive signaling pathway having various roles in the normal origin and development of each multicellular organisms. Therefore, any aberration in the pathway will inevitably lead to deadly outcomes such as cancer. It has now been more than two decades since Notch was acknowledged as an oncogene in mouse mammary tumor virus-infected mice. Since that discovery, activated Notch signaling and consequent up-regulation of tumor-promoting Notch target genes have been observed in human breast cancer. Moreover, consistent over-expression of Notch ligands and receptors has been shown to correlate with poor prognosis in human breast cancer. Notch regulates a number of key processes during breast carcinogenesis, of which, one key phenomenon is epithelial–mesenchymal transition (EMT). EMT is a key process for large-scale cell movement during morphogenesis at the time of embryonic development. Cancer cells aided by transcription factors usurp this developmental program to execute the multi-step process of tumorigenesis and metastasis. In this review, we recapitulate recent progress in breast cancer research that has provided new perceptions into the molecular mechanisms behind Notch-mediated EMT regulation during breast tumorigenesis.


1999 ◽  
Vol 10 (7) ◽  
pp. 2329-2342 ◽  
Author(s):  
Beth E. Stronach ◽  
Patricia J. Renfranz ◽  
Brenda Lilly ◽  
Mary C. Beckerle

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis ofDrosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present inMlp genomic sequences. These data suggest that theMlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and α-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.


2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Qi Zhu ◽  
Feng Liang ◽  
Shufang Cai ◽  
Xiaorong Luo ◽  
Tianqi Duo ◽  
...  

AbstractHistone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.


2021 ◽  
Author(s):  
Siyi Xie ◽  
Chushan Fang ◽  
Yujie Gao ◽  
Jie Yan ◽  
Lina Luo ◽  
...  

Abstract Background: Skeletal muscle is composed of bundles of myofibers ensheathed by extracellular matrix networks. Malformation of skeletal muscle during embryonic development results in congenital myopathies. Disease mechanisms of congenital myopathies remain unclear. PINCH, an adaptor of focal adhesion complex, plays essential roles in multiple cellular processes and organogenesis. Elucidation of the molecular mechanisms underlying skeletal myogenesis will offer new insights into pathogenesis of myopathies.Methods: We generated muscle-specific PINCH knock-out mice to study the functional role of PINCH in skeletal myogenesis. Histologic and Transmission Electron Microscopy analysis demonstrated that Impaired myogenic differentiation and maturation in mice with PINCH1 being ablated in skeletal muscle progenitors, and Ablation of PINCH1 and PINCH2 resulted in reduced size of muscle fibers and impaired multinucleation; Cell culture and immunostaining showed that defects in myoblast fusion and cytoskeleton assembly in PINCH double mutant mice; Western blotting showed that defects in expression of cytoskeleton proteins and proteins involved in myogenesis in DMUT skeletal muscles.Results: Double ablation of PINCH1 and PINCH2 resulted in early postnatal lethality with reduced size of skeletal muscles and detachment of diaphragm muscles from the body wall. Myofibers of PINCH mutant myofibers failed to undergo multinucleation and exhibited disrupted sarcomere structures. The mutant myoblasts in culture were able to adhere to newly formed myotubes, but impeded in cell fusion and subsequent sarcomere genesis and cytoskeleton organization. Consistent with this, expression of integrin β1 and some cytoskeleton proteins, and phosphorylation of ERK and AKT were significantly reduced in PINCH mutants. Expression of MRF4, the most highly expressed myogenic factor at late stages of myogenesis, was abolished in PINCH mutants, that could contribute to observed phenotypes. In addition, mice with PINCH1 being ablated in myogenic progenitors exhibited only mild centronuclear myopathic changes, suggesting a compensatory role of PINCH2 in myogenic differentiation, indicating a critical role of PINCH proteins in myogenic differentiation.Conclusion: Our results demonstrated an essential role of PINCH in skeletal myogenic differentiation.


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