Induction of myogenic differentiation by an expression vector encoding the DNA methyltransferase cDNA sequence in the antisense orientation.

To test the hypothesis that DNA methylation controls the state of differentiation of a mammalian cell, we transfected the stable mesenchymal line 10T1/2 with an expression vector encoding sequences from the DNA methyltransferase (DNA MeTase) cDNA in the antisense orientation. 10T1/2 cells transfected with the antisense construct (pZ alpha M), but not with the vector alone, exhibit morphological changes, convert into multinucleated tubular cells, and express the skeletal myosin heavy chain protein. The conversion to myogenic phenotype is a late event and is dependent on the number of replication events that the cell has undergone, suggesting that induction of myogenesis is a multistep process. Demethylation of sequences that are not involved in the myogenic process is detected at early passages, while demethylation and expression of the MyoD gene is a late event. This report establishes for the first time that demethylation is a very early event in commitment to myogenic differentiation, while demethylation and expression of MyoD is a late event. We suggest that other genes serve as the initial targets for demethylation and commitment of mesenchymal cells to myogenesis. The cell lines described in this report can serve as an important system for identifying these genes.     

Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1

Myogenin is required not for the initiation of myogenesis but instead for skeletal muscle formation through poorly understood mechanisms. We demonstrate in cultured cells and, for the first time, in embryonic tissue, that myogenic late genes that specify the skeletal muscle phenotype are bound by MyoD prior to the initiation of gene expression. At the onset of muscle specification, a transition from MyoD to myogenin occurred at late gene loci, concomitant with loss of HDAC2, the appearance of both the Mef2D regulator and the Brg1 chromatin-remodeling enzyme, and the opening of chromatin structure. We further demonstrated that ectopic expression of myogenin and Mef2D, in the absence of MyoD, was sufficient to induce muscle differentiation in a manner entirely dependent on Brg1. These results indicate that myogenin specifies the muscle phenotype by cooperating with Mef2D to recruit an ATP-dependent chromatin-remodeling enzyme that alters chromatin structure at regulatory sequences to promote terminal differentiation.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

Myostatin通过Smad3下调MyoD的表达来抑制骨骼肌卫星细胞的分化

Myostatin基因,是肌肉生长的负调控因子,通过下调MyoD的表达抑制骨骼肌细胞的分化,但具体机制目前尚未完全清楚。本研究以体外培养的猪骨骼肌卫星细胞为实验材料,利用RNAi 技术,以Smad3为靶基因进行干扰研究,研究干扰前后猪骨骼肌卫星细胞增殖情况的变化以及MyoD、Myostatin基因的表达规律,进一步阐述三个基因间的调控关系。结果表明,Myostatin通过下调MyoD的表达,抑制骨骼肌卫星细胞的分化,但这种抑制作用是受Smad3调节的。     

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.