Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.     

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.     

Have giant lobelias evolved several times independently? Life form shifts and historical biogeography of the cosmopolitan and highly diverse subfamily Lobelioideae (Campanulaceae)

Background

Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation.

Results

Treatment of P19 or mouse ES cells with low levels of RA led to an enhancement of skeletal myogenesis by upregulating the expression of the mesodermal marker, Wnt3a, the skeletal muscle progenitor factors Pax3 and Meox1, and the myogenic regulatory factors (MRFs) MyoD and myogenin. By chromatin immunoprecipitation, RA receptors (RARs) bound directly to regulatory regions in the Wnt3a, Pax3, and Meox1 genes and RA activated a β-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative β-catenin/engrailed repressor fusion protein, RA could not bypass the inhibition of skeletal myogenesis nor upregulate Meox1 or MyoD. Thus, RA functions both upstream and downstream of Wnt signalling. In contrast, it functions downstream of BMP4, as it abrogates BMP4 inhibition of myogenesis and Meox1, Pax3, and MyoD expression. Furthermore, RA downregulated BMP4 expression and upregulated the BMP4 inhibitor, Tob1. Finally, RA inhibited cardiomyogenesis but not in the presence of BMP4.

Conclusion

RA can enhance skeletal myogenesis in stem cells at the muscle specification/progenitor stage by activating RARs bound directly to mesoderm and skeletal muscle progenitor genes, activating β-catenin function and inhibiting bone morphogenetic protein (BMP) signalling. Thus, a signalling pathway can function at multiple levels to positively regulate a developmental program and can function by abrogating inhibitory pathways. Finally, since RA enhances skeletal muscle progenitor formation, it will be a valuable tool for designing future stem cell therapies.     

Myogenin regulates a distinct genetic program in adult muscle stem cells

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.     

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.     

成肌调节因子MyoD和myogenin在不同月龄DMD模型鼠mdx小鼠的表达

目的探讨成肌调节因子MyoD和myogenin在不同月龄DMD模型鼠mdx鼠的表达情况。方法取不同月龄DMD模型鼠mdx鼠以及相应的同龄正常C57鼠的腓肠肌,冰冻切片后用HE染色显示肌肉病理,SABC-DAB染色检测成肌调节因子MyoD和myogenin的表达。结果不同月龄mdx鼠肌肉坏死和再生程度不同,MyoD和myogenin在1月龄mdx鼠表达最强,在13月龄mdx鼠仍有表达,在正常同龄C57鼠不表达。结论MyoD与Myogenin在肌肉损伤后的再生修复过程中起作用,可作为鉴定肌肉前体细胞和反映肌肉再生的指标。     

Effects of 17β-estradiol on turkey myogenic satellite cell proliferation,differentiation, and expression of glypican-1, MyoD and myogenin

The hypothesis of this study was that 17β-estradiol (estradiol) stimulates turkey skeletal muscle growth by influencing myogenic satellite cell proliferation, differentiation, and the gene expression of selected proteins important in regulating growth and development. Increasing levels of estradiol were administered in basal medium containing additional nutrients. Female-derived pectoralis major (PM) satellite cell proliferation was stimulated by estradiol at a level of 10^? 9 M following 4 days of treatment. Male PM and biceps femoris (BF) satellite cell proliferation was increased at 10^? 12 M estradiol. Turkey embryonic myoblast proliferation, however, decreased with 10^? 9 M and 10^? 5 M estradiol following 3 days under these conditions. Estradiol had no effect on the differentiation of any of the 4 groups of cells. Likewise, glypican-1 expression was unaffected by estradiol treatment. MyoD expression decreased in male PM but not BF cells. MyoD expression in female PM cells and embryonic myoblasts were also unaffected by estradiol administration. Estradiol decreased myogenin expression in male satellite cells, but had no effect on female cells. There was a slight decrease in myogenin expression in embryonic myoblasts. The results demonstrate a direct effect of estradiol on avian satellite cell proliferation independent of glypican-1, and decreased expression of MyoD and myogenin in some myogenic cells, coinciding with increased cellular proliferation.     

MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells

myogenin (-/-) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene expression. To distinguish between these two possibilities, we compared the abilities of constitutively expressed myogenin and MyoD to support muscle differentiation in embryoid bodies made from myogenin (-/-) ES cells. Differentiated embryoid bodies from wild-type embryonic stem (ES) cells made extensive skeletal muscle, but embryoid bodies from myogenin (-/-) ES cells had greatly attenuated muscle-forming capacity. The inability of myogenin (-/-) ES cells to generate muscle was independent of endogenous MyoD expression. Skeletal muscle was restored in myogenin (-/-) ES cells by constitutive expression of myogenin. In contrast, constitutive expression of MyoD resulted in only marginal enhancement of skeletal muscle, although myocyte numbers greatly increased. The results indicated that constitutive expression of MyoD led to enhanced myogenic commitment of myogenin (-/-) cells but also indicated that committed cells were impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in their expression properties.