Showing papers on "MYF5 published in 2002"

Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos.

Abstract: Embryological and genetic studies of mouse, bird, zebrafish, and frog embryos are providing new insights into the regulatory functions of the myogenic regulatory factors, MyoD, Myf5, Myogenin, and MRF4, and the transcriptional and signaling mechanisms that control their expression during the specification and differentiation of muscle progenitors. Myf5 and MyoD genes have genetically redundant, but developmentally distinct regulatory functions in the specification and the differentiation of somite and head muscle progenitor lineages. Myogenin and MRF4 have later functions in muscle differentiation, and Pax and Hox genes coordinate the migration and specification of somite progenitors at sites of hypaxial and limb muscle formation in the embryo body. Transcription enhancers that control Myf5 and MyoD activation in muscle progenitors and maintain their expression during muscle differentiation have been identified by transgenic analysis. In epaxial, hypaxial, limb, and head muscle progenitors, Myf5 is controlled by lineage-specific transcription enhancers, providing evidence that multiple mechanisms control progenitor specification at different sites of myogenesis in the embryo. Developmental signaling ligands and their signal transduction effectors function both interactively and independently to control Myf5 and MyoD activation in muscle progenitor lineages, likely through direct regulation of their transcription enhancers. Future investigations of the signaling and transcriptional mechanisms that control Myf5 and MyoD in the muscle progenitor lineages of different vertebrate embryos can be expected to provide a detailed understanding of the developmental and evolutionary mechanisms for anatomical muscles formation in vertebrates. This knowledge will be a foundation for development of stem cell therapies to repair diseased and damaged muscles.

Myf5 is a direct target of long-range Shh signaling and Gli regulation for muscle specification.

Abstract: Sonic hedgehog (Shh) is a secreted signaling molecule for tissue patterning and stem cell specification in vertebrate embryos. Shh mediates both long-range and short-range signaling responses in embryonic tissues through the activation and repression of target genes by its Gli transcription factor effectors. Despite the well-established functions of Shh signaling in development and human disease, developmental target genes of Gli regulation are virtually unknown. In this study, we investigate the role of Shh signaling in the control of Myf5, a skeletal muscle regulatory gene for specification of muscle stem cells in vertebrate embryos. In previous genetic studies, we showed that Shh is required for Myf5 expression in the specification of dorsal somite, epaxial muscle progenitors. However, these studies did not distinguish whether Myf5 is a direct target of Gli regulation through long-range Shh signaling, or alternatively, whether Myf5 regulation is a secondary response to Shh signaling. To address this question, we have used transgenic analysis with lacZ reporter genes to characterize an Myf5 transcription enhancer that controls the activation of Myf5 expression in the somite epaxial muscle progenitors in mouse embryos. This Myf5 epaxial somite (ES) enhancer is Shh-dependent, as shown by its complete inactivity in somites of homozygous Shh mutant embryos, and by its reduced activity in heterozygous Shh mutant embryos. Furthermore, Shh and downstream Shh signal transducers specifically induce ES enhancer/luciferase reporters in Shh-responsive 3T3 cells. A Gli-binding site located within the ES enhancer is required for enhancer activation by Shh signaling in transfected 3T3 cells and in epaxial somite progenitors in transgenic embryos. These findings establish that Myf5 is a direct target of long-range Shh signaling through positive regulation by Gli transcription factors, providing evidence that Shh signaling has a direct inductive function in cell lineage specification.

Limb muscle development.

Abstract: Skeletal muscle precursors for the limbs originate from the epithelial layer of the somites, the dermomyotomes. We summarize the steps of limb muscle development from the specification of precursor cells in the dermomyotome, the directed migration of these cells to and within the limb buds to muscle growth and differentiation. All steps are controlled by local signaling between embryonic structures. In dermomyotome development, signals from the neural tube, the ectoderm and the intermediate and lateral mesoderm result in a medio-lateral patterning. Only the lateral portions of the dermomyotomes give rise to muscle precursor cells destined to enter the limb buds. As a prerequisite for migration, precursor cells have to de-epithelialize as a result of interactions between SF/HGF and its receptor c-met. Precursor cells adopt a mesenchymal morphology without losing their myogenic specification. This is achieved by the expression of the transcription factors Pax3, Pax7 and myf5. During migration, premature differentiation has to be kept at bay to enable motility and proliferation. After having reached their target sites, the dorsal and ventral myogenic zones, myogenesis is initiated by the activation of the muscle determination factors MyoD, myogenin and MRF4. Finally, we briefly summarize the process of muscle hypertrophy and regeneration during which aspects of developmental processes are reinitiated.

FGFR4 signaling is a necessary step in limb muscle differentiation.

Abstract: In chick embryos, most if not all, replicating myoblasts present within the skeletal muscle masses express high levels of the FGF receptor FREK/FGFR4, suggesting an important role for this molecule during myogenesis. We examined FGFR4 function during myogenesis, and we demonstrate that inhibition of FGFR4, but not FGFR1 signaling, leads to a dramatic loss of limb muscles. All muscle markers analyzed (such as Myf5, MyoD and the embryonic myosin heavy chain) are affected. We show that inhibition of FGFR4 signal results in an arrest of muscle progenitor differentiation, which can be rapidly reverted by the addition of exogenous FGF, rather than a modification in their proliferative capacities. Conversely, over-expression of FGF8 in somites promotes FGFR4 expression and muscle differentiation in this tissue. Together, these results demonstrate that in vivo, myogenic differentiation is positively controlled by FGF signaling, a notion that contrasts with the general view that FGF promotes myoblast proliferation and represses myogenic differentiation. Our data assign a novel role to FGF8 during chick myogenesis and demonstrate that FGFR4 signaling is a crucial step in the cascade of molecular events leading to terminal muscle differentiation.

Involvement of myogenic regulator factors during fusion in the cell line C2C12.

Abstract: The myogenic factors, MyoD, myogenin, Myf5 and MRF4, can activate skeletal muscle differentiation when overexpressed in non-muscular cells Gene targeting experiments have provided much insight into the in vivo functions of MRF and have defined two functional groups of MRFs MyoD and Myf5 may be necessary for myoblast determination while myogenin and MRF4 may be required later during differentiation However, the specific role of these myogenic factors has not been clearly defined during one important stage of myogenesis: the fusion of myoblasts Using cultured C2C12 mouse muscular cells, the time-course of these proteins was analyzed and a distinct expression pattern in fusing cells was revealed In an attempt to clarify the role of each of these regulators during myoblast fusion, an antisense strategy using oligonucleotides with phosphorothioate backbone modification was adoped The results showed that the inhibition of myogenin and Myf5 activity is capable of significantly preventing fusion Furthermore, the inhibition of MyoD can wholly arrest the engaged fusion process in spite of high endogenous expression of both myogenin and Myf5 Consequently, each MRF seems to have, at this defined step of myogenesis, a specific set of functions that can not be substituted for by the others and therefore may regulate a distinct subset of muscle-specific genes at the onset of fusion

Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury.

Abstract: Myogenic precursor cells known as satellite cells persist in adult skeletal muscle and are responsible for its ability to regenerate after injury. Quiescent satellite cells are activated by signals emanating from damaged muscle. Here we describe the rapid activation of two genes in response to muscle injury; these transcripts encode LPS-inducible CXC chemokine (LIX), a neutrophil chemoattractant, and Tristetraprolin (TTP), an RNA-binding protein implicated in the regulation of cytokine expression. Using a synchronized cell culture model we show that C2C12 myoblasts arrested in G0 exhibit some molecular attributes of satellite cells in vivo: suppression of MyoD and Myf5 expression during G0 and their reactivation in G1. Synchronization also revealed cell cycle dependent expression of CD34, M-cadherin, HGF and PEA3, genes implicated in satellite cell biology. To identify other genes induced in synchronized C2C12 myoblasts we used differential display PCR and isolated LIX and TTP cDNAs. Both LIX and TTP mRNAs are short-lived, encode molecules implicated in inflammation and are transiently induced during growth activation in vitro. Further, LIX and TTP are rapidly induced in response to muscle damage in vivo. TTP expression precedes that of MyoD and is detected 30 minutes after injury. The spatial distribution of LIX and TTP transcripts in injured muscle suggests expression by satellite cells. Our studies suggest that in addition to generating new cells for repair, activated satellite cells may be a source of signaling molecules involved in tissue remodeling during regeneration.

The early epaxial enhancer is essential for the initial expression of the skeletal muscle determination gene Myf5 but not for subsequent, multiple phases of somitic myogenesis

Abstract: Vertebrate myogenesis is controlled by four transcription factors known as the myogenic regulatory factors (MRFs): Myf5, Mrf4, myogenin and MyoD. During mouse development Myf5 is the first MRF to be expressed and it acts by integrating multiple developmental signals to initiate myogenesis. Numerous discrete regulatory elements are involved in the activation and maintenance of Myf5 gene expression in the various muscle precursor populations, reflecting the diversity of the signals that control myogenesis. Here we focus on the enhancer that recapitulates the first phase of Myf5 expression in the epaxial domain of the somite, in order to identify the subset of cells that first transcribes the gene and therefore gain insight into molecular, cellular and anatomical facets of early myogenesis. Deletion of this enhancer from a YAC reporter construct that recapitulates the Myf5 expression pattern demonstrates that this regulatory element is necessary for expression in the early epaxial somite but in no other site of myogenesis. Importantly, Myf5 is subsequently expressed in the epaxial myotome under the control of other elements located far upstream of the gene. Our data suggest that the inductive signals that control Myf5 expression switch rapidly from those that impinge on the early epaxial enhancer to those that impinge on the other enhancers that act later in the epaxial somite, indicating that there are significant changes in either the signalling environment or the responsiveness of the cells along the rostrocaudal axis. We propose that the first phase of Myf5 epaxial expression, driven by the early epaxial enhancer in the dermomyotome, is necessary for early myotome formation, while the subsequent phases are associated with cytodifferentiation within the myotome.

Six and Eya expression during human somitogenesis and MyoD gene family activation.

Abstract: This report describes the characterisation of the expression profile of several myogenic determination genes during human embryogenesis. The data were obtained from axial structures and limb buds of human embryos aged between 3 and 8 weeks of development. Using in situ hybridisation to detect Pax3 and MyoD gene family mRNAs, and immunochemistry to follow Six and Eya protein accumulation, we have been able to establish the chronology of accumulation of these gene products. As in mouse, the first transcripts detected in myotomes of 3 week-old embryos are Pax3 and Myf5, followed by the expression of myogenin. MyoD appears to be activated well after Myf5, myogenin and MRF4 in the early myotome, whereas, in limb bud muscles, the presence of all four of these mRNAs is concomitant from 6 weeks. Six1, Six4 and Six5 homeoproteins are detected later than Myf5 activation. These Six homeoproteins are first observed in the cytoplasm of myogenin expressing cells. At later stages of development, Six1 and Six5, but not Six4, are translocated into the nuclei of myogenic cells, concomitantly with MyHCemb expression. Eya1 and Eya2 proteins, potential Six cofactors, were also detected in myogenin positive cells, but their accumulation was delayed and was mainly cytoplasmic. These results preclude that early activation of Myf5, myogenin and MRF4 is under the control of Six and Eya proteins, while Six and Eya proteins would be involved in later steps of myogenic differentiation.

Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream ( Sparus aurata).

Abstract: Members of the myogenic regulatory gene family, including MyoD, Myf5, Myogenin and MRF4, are specifically expressed in myoblast and skeletal muscle cells and play important roles in regulating skeletal muscle development and growth. They are capable of converting a variety of non-muscle cells into myoblasts and myotubes. To better understand their roles in the development of fish muscles, we have isolated the MyoD genomic genes from gilthead seabream (Sparus aurata), analyzed the genomic structures, patterns of expression and the regulation of muscle-specific expression. We have demonstrated that seabream contain two distinct non-allelic MyoD genes, MyoD1 and MyoD2. Sequence analysis revealed that these two MyoD genes shared a similar gene structure. Expression studies demonstrated that they exhibited overlapping but distinct patterns of expression in seabream embryos and adult slow and fast muscles. MyoD1 was expressed in adaxial cells that give rise to slow muscles, and lateral somitic cells that give rise to fast muscles. Similarly, MyoD2 was initially expressed in both slow and fast muscle precursors. However, MyoD2 expression gradually disappeared in the adaxial cells of 10- to 15-somite-stage embryos, whereas its expression in fast muscle precursor cells was maintained. In adult skeletal muscles, MyoD1 was expressed in both slow and fast muscles, whereas MyoD2 was specifically expressed in fast muscles. Treating seabream embryos with forskolin, a protein kinase A activator, inhibited MyoD1 expression in adaxial cells, while expression in fast muscle precursors was not affected. Promoter analysis demonstrated that both MyoD1 and MyoD2 promoters could drive green fluorescence protein expression in muscle cells of zebrafish embryos. Together, these data suggest that the two non-allelic MyoD genes are functional in seabream and their expression is regulated differently in fast and slow muscles. Hedgehog signaling is required for induction of MyoD expression in adaxial cells.

Related expression of MyoD and Myf5 with myosin heavy chain isoform types in bovine adult skeletal muscles.

Abstract: Skeletal muscles are characterized as fast and slow muscles, according to the expression pattern of myosin heavy chain (MyHC) isoforms in the muscle fibers. To investigate the relationships between MyHC isoforms and myogenic regulatory factors (MRFs) including MyoD, Myf5, myogenin, and MRF4 in adult skeletal muscles, expressions of these MRFs in the ten muscles of three cows were analyzed by a semi-quantitative RT-PCR. The results showed that MyoD expression was significantly lower in the lingual muscles (TN), masseter (MS) and diaphragm (DP), which lack MyHC-2x (fast glycolytic) expression and abound with MyHC-slow (slow oxidative) and/or MyHC-2a (fast oxidative), than it was in the pectoralis (PP), psoas major (PM), longissimus thoracis (LT), spinnalis (SP), semitendinosus (ST), semimembranosus (SM), and biceps femoris (BF). In contrast, the Myf5 expression in TN, MS, and DP was significantly higher than in PM, LT, ST, SM, and BF. No significant difference was observed in myogenin and MRF4 expression among the muscles tested. The results suggest that MyoD and Myf5 influence the MyHC isoform expression, although the effects are not decisive in specifying the phenotypes of adult muscles.

Mammalian smooth muscle differentiation: origins, markers and transcriptional control.

Abstract: Three principal muscle types have evolved in essentially all vertebrate species to carry out functions related to cellular contraction. Traditionally, the three muscle types — cardiac, skeletal and smooth — have been distinguished by their unique structural and functional attributes. In recent years, great strides have been made with respect to the molecular characteristics of each muscle type and the regulatory pathways and factors governing muscle cell lineage determination and differentiation. This is especially true with skeletal and cardiac muscle where several transcription factors have been assigned critical roles in orchestrating developmental programmes unique to these two sarcomeric muscle types. The myogenic regulatory factors in skeletal muscle (myf5, MyoD, myogenin, and MRF4) have, indeed, formed the basis of a paradigm of cellular differentiation (Olson 1990). This paradigm has subsequently been extended to cardiac muscle where related transcription factors (e.g., dHAND) have been cloned and shown, through genetic means, to be essential for normal cardiogenesis (Olson and Srivastava 1996). The relative ease in which progress has been made with the two sarcomeric muscle types probably reflects their wellcircumscribed points of origin and their terminal differentiation.

The α-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor myogenin

Abstract: SPECIFIC AIMSRPB3 is a RNA polymerase II (pol II) α-like core subunit. Here we show that RPB3 directly contacts the myogenic transcription factor Myogenin, mediating in this way Myogenin/pol II interaction. RPB3 contacts only Myogenin; it does not bind the other myogenic factors such as MyoD, Myf5, and MRF4. These findings coincide with the Myogenin role played principally in differentiation/maintenance of the myogenic phenotype rather than in muscle determination. The interaction between RPB3 and Myogenin suggests a direct link between a tissue-specific transcription factor and pol II and appears to be critical for the ‘active’ maintenance of muscle differentiation state.PRINCIPAL FINDINGS1. RPB3 interacts with MyogeninTo identify protein/s interacting with RPB3, yeast two-hybrid experiments were performed using a cDNA library prepared from human skeletal muscle. Of 30 positive clones analyzed, two independent clones encoded for the basic helix loop helix (bHLH) myogenic transcription factor Myogenin. To...

Characterization of muscle-regulatory genes, Myf5 and myogenin, from striped bass and promoter analysis of muscle-specific expression.

Abstract: Myf5 and Myogenin are basic helix-loop-helix transcription factors that belong to the muscle regulatory factor (MRF) gene family, which plays important roles in regulating skeletal muscle development and growth. Members of the MRF family, including Myf5, MyoD, Myogenin, and MRF4 are specifically expressed in skeletal muscle cells. They have the remarkable property of converting a variety of cells into myoblasts and myotubes when ectopically expressed in other cell types. To better understand their role and regulation of expression in fish muscle cells, Myf5 and myogenin genomic genes were isolated from striped bass (Morone saxatilis). Sequence analysis revealed that these 2 genes shared similar structures. They both contained 3 exons and 2 introns, and a highly conserved basic helix-loop-helix domain. Promoter analysis identified several putative E box sites in both Myf5 and myogenin promoters that might confer muscle-specific expression. To determine if the striped bass Myf5 and myogenin promoters could control muscle-specific expression, the Myf5 or myogenin promoter was linked with the green fluorescent protein (GFP) reporter gene, and their promoter activity was analyzed in zebrafish embryos by transient expression assay. Our data showed that both striped bass Myf5 and myogenin promoters could drive muscle-specific GFP expression in zebrafish. These data demonstrated that a muscle-specific regulatory element or elements were located within the striped bass Myf5 and myogenin promoters, and were conserved between striped bass and zebrafish. Moreover, these data suggested the muscle-specific regulatory element could function across fish species in regulating gene expression.

16 Wnt regulation of limb muscle differentiation

Abstract: The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax-3 and are uncommitted to myogenic differentiation, but upon entering the limb field they become committed switching on the expression of MyoD and Myf5. The myogenic cells subsequently undergo terminal differentiation into slow or fast fibre types which have distinct contractile properties determining how a muscle will function. In general, fast fibre types contract rapidly with high force and are needed for movement whilst slow fibre types contract slowly and are largely required for maintenance of posture. During migration, and in the limb bud, the myogenic cells come within range of Wnt signals. Here we have investigated the role of Wnt signalling in the developing chick limb by gain- and loss-of-function studies in vitro and in vivo. We show that Wnt-3a and the Wnt antagonist Sfrp-2 reduce the number of terminally differentiated cells whilst Wnt-7a and -14 have the converse effect. Wnt signalling also changes the number of fast and/or slow fibre types: Wnt-11 decreases and increases the number of slow and fast fibre types respectively whilst Wnt-5a and -6 have the opposite effect. Therefore Wnt signalling is essential for the formation of the limb musculature controlling both the number of terminally differentiated myogenic cells and the ratio of slow and fast fibres determining the intricate patterning of the limb musculature.

Sequential expression of myogenic regulatory factors in bovine skeletal muscle and the satellite cell culture

Abstract: Myogenic regulatory factors (MRFs) are important in the control of skeletal muscle development. To understand myogenic regulation by MRFs in bovine adult muscle cells, their expressions, namely that of Myf5, MyoD, myogenin, and MRF4 in the biceps femoris muscle (BF) and in the satellite cell culture, were analyzed by RT-PCR. In the BF, all four MRFs were expressed and in particular, myogenin and MRF4 were strongly expressed, whereas Myf5 was faintly expressed. The satellite cells prepared from the BF expressed Myf5, but only a trace of MyoD, at day 9 of culture. During the growth of the cells to day 14, the MyoD and myogenin expressions gradually increased, and that of MyoD expression reached its maximum at the confluence of the culture. After induction of myogenic differentiation by a serum-free medium at day 14, Myf5 expression gradually decreased, and the up-regulated expression of MyoD was suppressed, whereas myogenin expression continued to increase sharply. Following the myogenin expression, MRF4 also drastically increased toward the myotube formation of the cells. When huge myotubes were formed at day 18, Myf5 was expressed at a low level, whereas the MyoD expression remained at a moderate level.

Different regulatory elements within the MyoD promoter control its expression in the brain and inhibit its functional consequences in neurogenesis.

Abstract: MyoD is a key basic helix-loop-helix (bHLH) transcription factor capable of converting many cells into skeletal muscle. Together with Myf5 it is essential for initiating skeletal myogenesis. In this report, the restricted domains of MyoD- lacZ expression have been revealed in the embryonic mouse brain by the analysis of transgenic mice with reporter genes driven by MyoD regulatory elements. The MD6.0-lacZ transgene was localized in the basal plate of pons, medulla oblongata (i.e. the medial longitudinal fasciculus) and spinal cord of wild-type and mutant mouse embryos at various stages of development, whereas the 258/−2.5lacZ transgene was not detected in the brain. In addition, MyoD RNA and MyoD protein accumulations were monitored in neurons expressing MD6.0-lacZ transgene. Although MyoD was detected in muscle myotomal cells, it was absent in MD6.0-lacZ -expressing neurons. This would account for the lack of myogenic conversion in brain structures and the absence of a neural phenotype in MyoD−/− embryos and mice. Together, these data indicate that within the promoter of MyoD different regulatory elements control its expression and prevent the functional consequences of MyoD in neurogenesis.

The genetics of murine skeletal muscle biogenesis.

Abstract: Skeletal muscle formation involves a complex interplay of cell movements, cell-cell signalling and the activation of key intracellular genes. In the mouse, the myogenic regulatory factors (MRFs) Myf5 and Myod, act as the gatekeepers into the skeletal muscle lineage. Since this entry point into myogenesis was described, more interest has focused on the immediate upstream and downstream events that control the establishment of the skeletal muscle programme. This review is centred on how certain key regulatory genes are involved in establishing skeletal muscles in the mouse embryo and some recent findings that have established skeletal myogenesis as an important paradigm for studying the restriction of cell fate during embryonic, fetal and postnatal development. Other aspects of myogenesis have been reviewed extensively in recent years, and the reader is invited to refer to them for more detailed information on the subject (Ordahl 2000 and references therein).

Chapter 1 The myogenic regulatory factors

Abstract: Publisher Summary This chapter focuses on the mycogenic regulatory factors. The chapter illustrates that the first hints to the existence of a dominant-acting myogenic transcription factor come from studies in which non-muscle cells were fused to myoblasts and muscle specific gene expression was activated in the non-muscle nuclei of the heterocaryons. This transcription factor has been identified by a subtractive cDNA approach and called MyoD. MyoD is a basic helix loop helix transcription factor capable of inducing myogenic gene expression in non-muscle cells. Myf5, Myogenin and MRF4 have been identified as members of the MyoD family of transcription factors, also called the myogenic regulator factors (MRFs). A similar capacity to induce muscle specific gene expression in non-muscle cells is demonstrated in the chapter for these MRFs. Members of this family have subsequently been identified in a diverse array of organisms including: Drosophila, Xenopus, Chicken, mouse, and human.