Control Of Muscle Differentiation Research Articles

Molecular and bioinformatic strategies for gene discovery for meat traits: a reverse genetics approach

The identification of genes that influence meat quality and meat yield has relevance for several livestock species. Candidate genes include those involved in the biochemical pathways controlling muscle differentiation, growth and development. Mutations in one such gene, myostatin, have previously been reported to have dramatic effects on muscle phenotype in cattle. Here we report a screening strategy for the discovery of novel mutations in 10 genes involved in muscle development using single-stand conformation polymorphism gels and DNA sequencing. Several novel mutations, both non-synonymous and synonymous were discovered, and some of these may alter gene function. In addition, we also conducted a meta-analysis of published quantitative trait loci from cattle, sheep, pigs and mice, identifying those muscle development genes most likely to contribute to variation in muscle traits within species. From this strategy we found several genes that map into regions that are part of the extended muscle development pathway.

Novel cis-element in intron 1 represses somite expression of zebrafish myf-5

Myf-5 is a basic helix-loop-helix (bHLH) transcription factor that controls muscle differentiation. During early embryogenesis, myf-5 expression is transient, somite- and stage-specific. However, the negative regulation of myf-5 is poorly understood. We constructed a plasmid [(−9977/−1)/E1/I1/E2/GFP] that contains the sequence −9977 to −1, exon 1 (E1), intron 1 (I1), and exon 2 (E2) of zebrafish ( Danio rerio) myf-5 and a reporter GFP gene. This plasmid was microinjected into zebrafish zygotes. Surprisingly, the somite-specific expression rate of reporter GFP in the transgenic embryos was extremely low (2%, n=392), compared to that of (−9977/−1)/GFP (92%, n=210). Dramatic repression of myf-5 expression was also observed in embryos microinjected with plasmids in which the sequence −8600/−1, −2937/−1 or −290/−1 was linked to E1/I1/E2/GFP. Thus, intron 1 contains a silencer that specifically represses the activity of myf-5. Functional analysis of intron 1 showed a strong, negative, cis-regulatory element was located at +502/+835. Its function was orientation- and position-dependent. The repressive capability of this silencer was completely dependent on two core motifs, IE1 (+502/+527) and IE2 (+816/+835), and a 156-bp spanning sequence that lies between them. This is the first study to identify a novel, cis-acting silencer in intron 1 that is crucial to negatively regulating zebrafish myf-5 expression.

Novel regulatory sequence -82/-62 functions as a key element to drive the somite-specificity of zebrafish myf-5.

Myf-5, a transcription factor that controls muscle differentiation, is expressed in somites during early embryogenesis. However, gene regulation of myf-5 is poorly understood and detailed functional analysis of the regulatory cis-elements is needed. In zebrafish, the myf-5 upstream sequence from -82 to -62 (-82/-62) was fused with a basal promoter and transferred to fertilized zebrafish eggs. The -82/-62 cassette drove green fluorescent protein (GFP) reporter gene expression specifically in the somites. Moreover, GFP signals were detected exclusively in the somites of 28-hpf embryos derived from eggs injected with pCMV-5x(-82/-62), which contained five copies of the -82/-62 cassette inserted within cytomegalovirus promoter/enhancer. Thus, the -82/-62 cassette, conserved in mouse myf-5, functions to drive somite-specific expression and to repress nonspecific expression during the early development of zebrafish embryos. Mutated sequence analysis of -82/-62 cassette showed that the -70/-62 sequence was the key element for controlling myf-5 specificity. The putative CCAAT-like box, located at -66/-62, could not direct somite-specific expression. A DNA-protein complex was specifically formed between the -70/-62 probe and embryonic nuclear extracts. We conclude that the -70/-62 motif is essential for controlling somite-specific expression and the CCAAT-like box is essential for activating gene transcription.

Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation.

Class II histone deacetylases (HDACs) 4, 5, 7, and 9 repress muscle differentiation through associations with the myocyte enhancer factor 2 (MEF2) transcription factor. MEF2-interacting transcription repressor (MITR) is an amino-terminal splice variant of HDAC9 that also potently inhibits MEF2 transcriptional activity despite lacking a catalytic domain. Here we report that MITR, HDAC4, and HDAC5 associate with heterochromatin protein 1 (HP1), an adaptor protein that recognizes methylated lysines within histone tails and mediates transcriptional repression by recruiting histone methyltransferase. Promyogenic signals provided by calcium/calmodulin-dependent kinase (CaMK) disrupt the interaction of MITR and HDACs with HP1. Since the histone methyl-lysine residues recognized by HP1 also serve as substrates for deacetylation by HDACs, the interaction of MITR and HDACs with HP1 provides an efficient mechanism for silencing MEF2 target genes by coupling histone deacetylation and methylation. Indeed, nucleosomal histones surrounding a MEF2-binding site in the myogenin gene promoter are highly methylated in undifferentiated myoblasts, when the gene is silent, and become acetylated during muscle differentiation, when the myogenin gene is expressed at high levels. The ability of MEF2 to recruit a histone methyltransferase to target gene promoters via HP1-MITR and HP1-HDAC interactions and of CaMK signaling to disrupt these interactions provides an efficient mechanism for signal-dependent regulation of the epigenetic events controlling muscle differentiation.

Insulin-like Growth Factor-mediated Muscle Differentiation: COLLABORATION BETWEEN PHOSPHATIDYLINOSITOL 3-KINASE-Akt-SIGNALING PATHWAYS AND MYOGENIN

The differentiation and maturation of skeletal muscle require interactions between signaling pathways activated by hormones and growth factors and an intrinsic regulatory network controlled by myogenic transcription factors. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo and in regeneration in the adult. To study mechanisms of IGF action in muscle, we developed a myogenic cell line that overexpresses IGF-binding protein-5. C2BP5 cells remain quiescent in low serum differentiation medium until the addition of IGF-I. Here we use this cell line to identify signaling pathways controlling IGF-mediated differentiation. Induction of myogenin by IGF-I and myotube formation were prevented by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, even when included 2 days after growth factor addition, whereas expression of active PI 3-kinase could promote differentiation in the absence of IGF-I. Differentiation also was induced by myogenin but was blocked by LY294002. The differentiation-promoting effects of IGF-I were mimicked by a modified membrane-targeted inducible Akt-1 (iAkt), and iAkt was able to stimulate differentiation of C2 myoblasts and primary mouse myoblasts incubated with otherwise inhibitory concentrations of LY294002. These results show that an IGF-regulated PI 3-kinase-Akt pathway controls muscle differentiation by mechanisms acting both upstream and downstream of myogenin.

Nerve control of type 2A MHC isoform expression in regenerating slow skeletal muscle.

Bupivacaine-induced regeneration was studied in rat soleus muscle under several conditions, with the focus on type 2A and type 1 myosin heavy chain (MHC) isoform expression. In denervated muscles, type 1 was absent, whereas type 2A was widely expressed, a pattern of regeneration which appeared to be independent of fibrillation activity of the muscle. Both type 1 and type 2A isoforms were absent in muscles regenerated during tetrodotoxin (TTX) block of impulse conduction in the sciatic nerve, but type 2A was still present when the TTX block was associated with the vinblastine block of axoplasmic flow; vinblastine block alone caused the coexpression of type 1 and type 2A isoforms in the majority of fibers. These results suggest that axoplasmic flow carries some chemical factor that inhibits 2A MHC isoform expression. The results are also of clinical interest, contributing to the understanding of factors controlling muscle differentiation and adaptation.

Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis.

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 of Drosophila 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 in Mlp genomic sequences. These data suggest that the Mlp 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 alpha-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.

P21(CIP1) and p57(KIP2) control muscle differentiation at the myogenin step.

Cell-cycle arrest is thought to be required for differentiation of muscle cells. However, the molecules controlling cell-cycle exit and the differentiation step(s) dependent on cell-cycle arrest are poorly understood. Here we show that two Cdk inhibitors, p21(CIP1) and p57(KIP2), redundantly control differentiation of skeletal muscle and alveoli in the lungs. Mice lacking both p21 and p57 fail to form myotubes, display increased proliferation and apoptotic rates of myoblasts, and display endoreplication in residual myotubes. This point of arrest during muscle development is identical to that of mice lacking the myogenic transcription factor myogenin, indicating a role for cell-cycle exit in myogenin function. Expression of myogenin, p21, and p57 is parallel but independent, and in response to differentiation signals, these proteins are coordinately regulated to trigger both cell-cycle exit and a dependent muscle-specific program of gene expression to initiate myoblast terminal differentiation and muscle formation.

IGFs and muscle differentiation.

The Role of IGFs in Myogenesis. Thus we are now convinced that the control of myogenesis by IGFs is a general phenomenon that occurs in all skeletal muscle cells, whether or not IGFs are added to the "differentiation" medium. We believe that several medium components contribute to the suppression of IGF-II expression in myoblasts incubated in high serum "growth" medium, and conclude that the IGF-I receptor mediates the feedback inhibition of IGF-II gene expression in muscle cells. Mechanism of Induction of Myogenesis by IGFs. The observations summarized here now permit a reasonably coherent overview of the stimulation of myogenic differentiation by the IGFs. It seems clear that all IGFs act by binding to the Type I IGF receptor, and that this process is inhibited to a significant extent by IGF binding proteins secreted by the target myoblasts. A major, but possibly not the only relevant effect of this binding is the induction of expression of the myogenin gene; this induction appears to require the presence of myf-5 protein, at least during the early part of the response. Cells capable of a mitogenic response undergo a round of division in response to IGF-I, thus delaying their entry into the final processes of postmitotic terminal differentiation. Other laboratories have shown that myogenin complexes with one or more widely occurring proteins such as E12 or E47 to form an active complex that interacts with CAnnTG elements in muscle specific genes, turning on expression of those genes and thus initiating the phenotype associated with terminally differentiated skeletal muscle.

Proto-oncogenes in the regulatory circuit for myogenesis

Skeletal muscle cells have provided an auspicious system for dissecting the mechanisms through which growth factor signals disrupt programs for cellular differentiation. Insight into the molecular mechanisms that control muscle differentiation has recently been obtained through the cloning of a family of muscle-specific transcription factors, often referred to as the MyoD family, that can activate myogenesis. The expression and activity of these factors are negatively regulated by growth factor signals and by activated oncogenes whose products transduce growth signals from the cell membrane to the nucleus. This review will focus on the role of proto-oncogenes in the transduction of growth factor signals that regulate myogenesis and on the cross-talk between the regulatory circuits that control myoblast proliferation and differentiation.

MyoD induces growth arrest independent of differentiation in normal and transformed cells.

MyoD is a gene involved in the control of muscle differentiation. We show that MyoD causes growth arrest when expressed in cell lines derived from tumors or transformed by different oncogenes. MyoD-induced growth inhibition was demonstrated by reduction in the efficiency of colony formation and at the single-cell level. We further show that MyoD growth inhibition can occur in cells that are not induced to activate muscle differentiation markers. The inhibitory activity of MyoD was mapped to the same 68-amino acid segment necessary and sufficient for induction of muscle differentiation, the basic-helix-loop-helix motif. Mutants with alterations in the basic region of MyoD that fail to bind or do not activate a muscle-specific enhancer inhibited growth; mutants with deletions in the helix-loop-helix region failed to inhibit growth. Thus, inhibition of cell growth by MyoD seems to occur by means of a parallel pathway to the one that leads to myogenesis. We conclude that MyoD is a prototypic gene capable of functionally activating intracellular growth inhibitory pathways.

Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype

Abstract Skeletal myoblasts from fetal muscle respond adversely to fibronectin and laminin substrata: When primary mouse skeletal myoblasts are plated onto laminin, more myosin and desmin-positive myoblasts (myo + cells) develop than on plates coated with fibronectin or collagen. In clonal cultures virtually all cells differentiate into postmitotic, fusion-capable myo+myoblasts on laminin after 3 days. In contrast, on fibronectin, the majority of the cells becomes myosin- and desmin-negative, partially due to proliferation of undifferentiated myoblast precursor cells, partially due to dedifferentiation or modulation of myoblasts into fibroblast-like myo - cells. Loss of the myogenic phenotype on fibronectin was also observed in cloned mouse myoblasts and in cultures of a differentiating mouse satellite cell line, MM14Dy, confirming that the appearance of desmin-negative cells is a result of myoblast modulation and not due simply to overgrowth by muscle fibroblasts. In the light of other effects of laminin on myoblasts, such as the stimulation of migration, differentiation and proliferation, our findings are consistent with the notion that laminin and fibronectin may be counteracting factors in the control of muscle differentiation.

Control of muscle differentiation in BC3H1 cells by fibroblast growth factor and vanadate.

We have examined the control of actin isoform synthesis by pituitary-derived fibroblast growth factor and serum in BC3H1 cells, a tumor-derived nonfusing muscle cell line. Under differentiating conditions in BC3H1 cells, the synthesis of beta- and gamma-actin ceases, and the rate of alpha-actin synthesis is increased concomitant with cessation of cell growth. Addition of fetal calf serum to differentiated cells reverses the process, whereas the addition of pituitary-derived fibroblast growth factor inhibits synthesis of alpha-actin but fails to induce the synthesis of beta- and gamma-actin. Analysis of RNA from differentiated BC3H1 cells after the addition of fetal calf serum indicated that the serum-induced increase in beta- and gamma-actin synthesis reflected an increase in their mRNA levels. In contrast, the repression of alpha-actin synthesis by fetal calf serum or fibroblast growth factor appears to reflect the translation efficiency of alpha-actin mRNA. Fibroblast growth factor is a competence factor for BC3H1 cells which allows them to progress from G0 4 h into the G1 phase of the cell cycle. In order to understand the nature of the intracellular signals responsible for the effect of fibroblast growth factor, we treated cells with vanadate, a known inhibitor of tyrosine-specific protein phosphatases. Vanadate fully mimics the action of fibroblast growth on actin synthesis and creatine phosphokinase synthesis and causes BC3H1 cells to exit the G0 portion of the cell cycle, as demonstrated by the induction of the c-fos proto-oncogene following addition of serum, vanadate, or bovine pituitary-derived fibroblast growth factor to these cells. We conclude that repression of alpha-actin synthesis and induction of the synthesis of beta- and gamma-actin are under independent control and that the induction of beta- and gamma-nonmuscle actin synthesis following serum addition is independent from movement into the cell cycle, and dependent on as yet unidentified serum components. The rate of synthesis of alpha-actin can be controlled by a defined mitogenic polypeptide fibroblast growth factor, which in short term experiments primarily affects the rate of translation of alpha-actin mRNA. The repression by fibroblast growth factor is most likely due to activation of a tyrosine specific protein kinase(s).

Control of muscle differentiation by embryonic neural tissues

ABSTRACT Previous work has suggested that in the salamander embryonic spinal cord enhances and embryonic brain suppresses differentiation of muscle from somitic mesoderm. These results have been confirmed by means of transplantation and explantation experiments performed on early neurulae of Ambystoma maculatum. Transplantation of presumptive spinal cord into the hind-brain region results in increased size of post-otic myotomes. On the other hand, transplantation of presumptive forebrain into the trunk neural plate usually results in a striking decrease in size of trunk myotomes. Orthotopic transplants of neural plate have little or no effect upon the development of myotomes. Ventral ectoderm transplanted into the neural plate at stage is incorporated into the host spinal cord and adjacent myotomes are usually normal. Transplants of trunk neural plate over lateral mesoderm often results in increased size of myotomes without affecting the pronephros. Explants of large amounts of somite mesoderm in an ectodermal envelope readily differentiate into muscle. Inclusion of presumptive brain cells in such explants results in a conspicuous decrease in frequency of occurrence of muscle differentiation and in quantity of muscle actually formed. The control of muscle differentiation in the early embryo is considered in the light of the known influences exerted by the neural tissues.