Phases Of Myogenesis Research Articles

Isolation and Culture of Muscle Stem Cells.

Polycomb group (PcG) proteins are key epigenetic factors responsible for the proper spatiotemporal repression of defined transcriptional programs along the process of cell differentiation, including myogenesis. The discovery of the pivotal role played by PcG factors during myogenic differentiation relied on the possibility to culture myogenic cells in vitro. We describe here the methods currently used to isolate muscle stem cells (MuSCs) both from single myofibers and from bulk muscles by fluorescence-activated cell sorting (FACS), highlighting experimental details and critical steps. Through these techniques MuSCs can be efficiently isolated and cultured in vitro to recapitulate the different phases of myogenesis: activation, expansion, differentiation, and self-renewal.

Thyroid hormones and skeletal muscle—new insights and potential implications

Thyroid hormone signalling regulates crucial biological functions, including energy expenditure, thermogenesis, development and growth. The skeletal muscle is a major target of thyroid hormone signalling. The type 2 and 3 iodothyronine deiodinases (DIO2 and DIO3, respectively) have been identified in skeletal muscle. DIO2 expression is tightly regulated and catalyses outer-ring monodeiodination of the secreted prohormone tetraiodothyronine (T4) to generate the active hormone tri-iodothyronine (T3). T3 can remain in the myocyte to signal through nuclear receptors or exit the cell to mix with the extracellular pool. By contrast, DIO3 inactivates T3 through removal of an inner-ring iodine. Regulation of the expression and activity of deiodinases constitutes a cell-autonomous, pre-receptor mechanism for controlling the intracellular concentration of T3. This local control of T3 activity is crucial during the various phases of myogenesis. Here, we review the roles of T3 in skeletal muscle development and homeostasis, with a focus on the emerging local deiodinase-mediated control of T3 signalling. Moreover, we discuss these novel findings in the context of both muscle homeostasis and pathology, and examine how skeletal muscle deiodinase activity might be therapeutically harnessed to improve satellite-cell-mediated muscle repair in patients with skeletal muscle disorders, muscle atrophy or injury.

Analysis of early C2C12 myogenesis identifies stably and differentially expressed transcriptional regulators whose knock-down inhibits myoblast differentiation

Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network regulating the later phases of myogenesis have been identified, relatively few studies have described the transcriptional landscape during the first 24 h, when myoblasts commit to differentiate. Through dense temporal profiling of differentiating C2C12 myoblasts, we identify 193 transcriptional regulators (TRs) whose expression is significantly altered within the first 24 h of myogenesis. A high-content shRNA screen of 77 TRs involving 427 stable lines identified 42 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. Of the TRs that were differentially expressed within the first 24 h, over half inhibited differentiation when knocked down, including known regulators of myogenesis (Myod1, Myog, and Myf5), as well as 19 TRs not previously associated with this process. Surprisingly, a similar proportion (55%) of shRNAs targeting TRs whose expression did not change also inhibited C2C12 myogenesis. We further show that a subset of these TRs inhibits myogenesis by downregulating expression of known regulatory and structural proteins. Our findings clearly illustrate that several TRs critical for C2C12 myogenesis are not differentially regulated, suggesting that approaches that focus functional studies on differentially-expressed transcripts will fail to provide a comprehensive view of this complex process.

N-cadherin and M-cadherin are sequentially expressed in myoblast populations contributing to the first and second waves of myogenesis in the trout (Oncorhynchus mykiss)

The objective of this study was to investigate the expression of two promyogenic cell surface adhesion receptors, N- and M-cadherin, in developing trout (Oncorhynchus mykiss) somite, taking account of the recent identification of a dermomyotome-like epithelium in teleosts. In situ hybridization showed that N-cadherin was expressed throughout the paraxial mesoderm and nascent somite. As the somite matured, N-cadherin expression disappeared ventrally from the sclerotome, and then mediolaterally from the differentiating slow and fast muscle cells of the embryonic myotome, to become finally restricted to the undifferentiated myogenic precursors forming the dermomyotome-like epithelium that surrounds the embryonic myotome. By contrast, M-cadherin, which was transcribed in the differentiating embryonic myotome, was never expressed in the dermomyotome-like epithelium. In late-stage trout embryos, M-cadherin transcript was only detected at the periphery of the expanding myotome, where muscle cells stemming from the N-cadherin positive dermomyotome-like epithelium differentiate. Collectively, our results support the view that, in trout embryo, N-cadherin is associated with muscle cell immaturity while M-cadherin is associated with muscle cell maturation and differentiation and this during the two successive phases of myogenesis.

A novel myogenic cell line with phenotypic properties of muscle progenitors

Skeletal myogenesis is a multistep process starting with progenitor cell proliferation, followed by their exit from the cell cycle, differentiation, alignment, and fusion to form multinucleated myotubes, typical of the differentiated muscle tissue. While the molecular players involved in early myogenesis have been extensively characterized, information about the later steps of the process is scanty. Here, we describe a novel myogenic cell line (MYOP7), composed of highly proliferating Sca-1+ muscle precursor cells, which can be induced to terminally differentiate into spontaneously contracting multinucleated myotubes. By performing high-density microarray analysis on these cells, we identified a series of genes, differentially expressed in proliferating vs differentiating conditions, which are candidates to play a major role in the later phase of myogenesis. In addition, we confirmed that the late stages of muscle differentiation are characterized by a marked upregulation of the cellular receptors for the vascular endothelial growth factor.

A key role ofPox mesoin somatic myogenesis ofDrosophila

The Pax gene Pox meso (Poxm) was the first and so far only gene whose initial expression was shown to occur specifically in the anlage of the somatic mesoderm, yet its role in somatic myogenesis remained unknown. Here we show that it is one of the crucial genes regulating the development of the larval body wall muscles in Drosophila. It has two distinct functions expressed during different phases of myogenesis. The early function, partially redundant with the function of lethal of scute [l(1)sc], demarcates the ;Poxm competence domain', a domain of competence for ventral and lateral muscle development and for the determination of at least some adult muscle precursor cells. The late function is a muscle identity function, required for the specification of muscles DT1, VA1, VA2 and VA3. Our results led us to reinterpret the roles of l(1)sc and twist in myogenesis and to propose a solution of the 'l(1)sc conundrum'.

Phases of myogenic cell activation and possible role of dermomyotome cells in teleost muscle formation

Present knowledge indicates that fibre recruitment (hyperplasia) in developing teleost fish occurs in three distinct phases. However, the origin and relationship of the myogenic precursors activated during the different phases remains unclear. Here, we address this issue using molecular techniques on embryos and larvae of pearlfish, a large cyprinid species. Results provide comprehensive molecular characterisation of cell recruitment over the three phases of myogenesis, identifying muscle types as they arise. Specifically, we show that the myogenic cells arising during 2nd phase myogenesis are clearly different from the myogenic cells arising during the 3rd phase and that the dermomyotome is a major source of myogenic cells driving 2nd phase hyperplasia. These findings are discussed in relation to their implications for the generality of vertebrate developmental patterns.

Post‐hatching development of the gut and lateral muscle in the sole

At hatching the gut of sole Solea solea was a primitive epithelial tube which showed vigorous cellular proliferation but only initial regionalization, and no secretory cells. By contrast, at this stage lateral muscle already consisted of a clearly distinct superficial monolayer of parvalbumin‐negative fibres, and a deeper layer of parvalbumin‐positive (presumptive fastwhite) fibres. There were no very small‐diameter fibres present and very few proliferating cell nuclear antigen (PCNA)‐positive nuclei, indicating that new fibres were not being produced at this time. The gut consisted of a distinct pharynx, oesophagus, stomach and intestine by 12 days, further differentiation of the anterior and posterior intestine was seen at 21 days, and morphological differentiation was complete at 2.5 months. Goblet cells containing acidic glycoconjugates were present at 12 days in the pharynx, oesophagus and anterior intestine, and increased in number at later ages. Neutral glycoconjugates first appeared in absorptive (not goblet) cells at 21 days. From 2.5 months neutral glycoconjugates were found in secretory cells throughout all parts of the gut except the stomach and the oesophagus, and by the adult stage the neutral glycoconjugates were the predominant type in the intestinal goblet cells. The difference in parvalbumin expression between the superficial (negative) and deep (positive) muscle layers seen at hatching persisted at all ages, and from 21 days immunostaining for slow isoforms of myosin gave a positive reaction in the superficial monolayer and subsequent slow muscle layer. Hypertrophic growth continued up to the largest subject examined, by posthatching myogenesis occurred in two distinct and relatively brief phases. The distribution of very small diameter muscle fibres and PCNA‐positive nuclei indicated that muscle hyperplasia occurred initially in apical areas of the myotomes (at 12 and 21 days), but this was followed (at 2.5 months) by a second ‘mosaic’ phase of myogenesis as new fibres appeared scattered throughout the deep muscle layer. Hyperplastic growth had ceased by 1 year.

Coordinate Regulation of Drosophila Tropomyosin Gene Expression Is Controlled by Multiple Muscle-Type-Specific Positive and Negative Enhancer Elements

Muscle development involves the coordinated regulation of transcription of muscle-type-specific genes and their encoded proteins during myogenesis. We show here that transcriptional regulation of the Drosophila tropomyosin I (TmI) gene during myogenesis is under the control of at least two muscle enhancer regions located within the first intron of the gene. Together these enhancer regions contain multiple muscle-type-specific positive and negative cis-acting elements which together contribute toward full expression of the gene. One of these enhancers is contained within a 355-bp fragment that is sufficient to direct high levels of temporally regulated expression from a heterologous promoter in all muscles of transgenic flies. Dissection of this enhancer region into smaller fragments has allowed us to identify a 91-bp enhancer fragment sufficient for directing expression in all somatic and visceral muscles of the larva and adult but not in the indirect flight muscles and tergal depressor of the trochanter or jump muscles of the adult. We also show that this somatic/visceral muscle element(s) can be repressed through an adjacent negative control region, suggesting that the regulation of expression in these muscles is under dual control during both phases of myogenesis. We propose a model in which transcriptional regulation of the Drosophila TmI gene is controlled by the cooperative interaction of multiple positive and negative cis-acting regulatory elements that control the temporal and muscle-type pattern of expression. The distribution of enhancer elements and their control of TmI gene expression are similar to those regulating transcription of the muscle promoter of the TmII gene and provide a framework for the coordinate expression of the two genes.

Ultrastructure and differentiation of ascidian muscle. II. Differentiation of the caudal muscle cells in the larva of Diplosoma macdonaldi.

The larval muscle cells of Diplosoma macdonaldi contain subcortical and medullary myofibrils which are invested by fenestrated sheets of the sarcoplasmic reticulum. Cisternae of the sarcoplasmic reticulum are coupled with tubular invaginations of the sarcolemma. To appreciate better such uncommon features of cellular organization, six embryonic stages were selected for an ultrastructural study of myogenesis. The proliferative, synthetic, and elaborative phases of myogenesis were represented by embryos ranging from neurulae to prehatching larvae. The contractile apparatus originates during the synthetic phase of myogenesis, when thick and thin myofilaments appear in the cortical sarcoplasm at the epidermal and notochordal poles of the cell. The myofilaments promptly aggregate into unstriated fascicles, and the fascicles unite in series to establish the rudimentary myofibrils. All major sarcomeric bands, except the Z-lines, are evident along the myofibrils. Cisternae of the sarcoplasmic reticulum form peripheral couplings with the overlying sarcolemma, and they also form interior couplings with sarcolemmal invaginations from the ends of the cell. The interior couplings localize over the I-bands of the myofibrils. In the elaborative phase of myogenesis, mitochondria invade the cortical sarcoplasm, and the contractile apparatus passively shifts to the subcortex and medulla of the cell. Relocation of the myofibrils coincides with the disappearance of all peripheral couplings. Cisternae of the sarcoplasmic reticulum anastomose around the myofibrils, creating the fenestrated sheets that extend between sarcomeres. As Z-lines begin to bisect the I-bands, the perifibrillar cisternae become confluent with the cisternae in the precocious interior couplings.