Expression of the cardiac actin gene in axolotl embryos

Axolotis are an important model system for studying heart development. Patterning of the somitic mesoderm occurs in axolotis in a manner that is much more similar to the pattern observed in higher vertebrates than in Xenopus. For these reasons we cloned the axolotl cardiac actin gene, since this gene is expressed during the development of both somitic and cardiac muscle in other vertebrates. In this paper we characterize its expression. Expression of cardiac actin RNA is switched on during gastrula stages and appears in the somitic mesoderm when it is formed; expression is later activated in the embryonic heart. In adults the gene is expressed only in the heart. The results demonstrate that the clone encoding cardiac actin provides a useful marker for studying development of both skeletal and cardiac muscle development in axolotls.     

Temporal resolution and sequential expression of muscle-specific genes revealed by in situ hybridization

The expression of muscle-specific mRNAs was analyzed directly within individual cells by in situ hybridization to chicken skeletal myoblasts undergoing differentiation in vitro. The probes detected mRNAs for sarcomeric myosin heavy chain (MHC) or the skeletal, cardiac, and beta isoforms of actin. Precise information as to the expression of these genes in individual cells was obtained and correlated directly with analyses of cell morphology and interactions, cell cycle stage, and immunofluorescence detection of the corresponding proteins. Results demonstrate that mRNAs for the two major muscle-specific proteins, myosin and actin, are not synchronously activated at the time of cell fusion. The mRNA for alpha-cardiac actin (CAct), known to be the predominant embryonic actin isoform in muscle, is expressed prior to cell fusion and prior to the expression of any isoform of muscle MHC mRNA. MHC mRNA accumulates rapidly immediately after fusion, whereas skeletal actin mRNA is expressed only in larger myofibers. Single cells expressing CAct mRNA have a characteristic short bipolar morphology, are in terminal G1, and do not contain detectable levels of the corresponding protein. In a pattern of expression reciprocal to that of CAct mRNA, beta-actin mRNA diminishes to low or undetectable levels in myofibers and in cells of the morphotype which expresses CAct mRNA. Finally, the intracellular distribution of mRNAs for different actin isoforms was compared using nonisotopic detection of isoform-specific oligonucleotide probes. This work illustrates a generally valuable approach to the analysis of cell differentiation and gene expression which directly integrates molecular, morphological, biochemical, and cell cycle information on individual cells.     

Myosin light chain-2 luciferase transgenic mice reveal distinct regulatory programs for cardiac and skeletal muscle-specific expression of a single contractile protein gene.

To examine the relationship between the cardiac and skeletal muscle gene programs, the current study employs the regulatory (phosphorylatable) myosin light chain (MLC-2) as a model system. Northern blotting, primer extension, and RNase protection studies documented the high level expression of the cardiac MLC-2 mRNA in both mouse cardiac and slow skeletal muscle (soleus). Transgenic mouse lines harboring a 2100- or a 250-base pair rat cardiac MLC-2 promoter/luciferase fusion gene were generated, demonstrating high levels of luciferase activity in cardiac muscle, and only background luminescence in slow skeletal muscle and non-muscle tissues. As assessed by in situ hybridization, immunofluorescence, and luminescence assays of luciferase reporter activity in various regions of the heart, both the endogenous MLC-2 gene and the MLC-2 luciferase fusion gene were expressed exclusively in the ventricular compartment, with expression in the atrium at background levels. Point mutations within the conserved regulatory sites HF-1a and HF-1b significantly cripple ventricular muscle specificity, while mutation of the single E-box site was without effect, suggesting that ventricular muscle-specific expression occurs through an E-box-independent pathway. This study provides direct evidence that the cis regulatory sequences in the cardiac/slow twitch MLC-2 gene which confer cardiac and skeletal muscle-specific expression can be clearly segregated, suggesting that distinct regulatory programs may have evolved to control the tissue-specific expression of this single contractile protein gene in cardiac and skeletal muscle.     

Tumor necrosis factor inhibits human myogenesis in vitro.

We examined the effects of human recombinant tumor necrosis factor-alpha (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of alpha-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as alpha-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon beta-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.     

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

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 MyoDgenes, 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 MyoDexpression in adaxial cells.     

Transgenic mice that express Cre recombinase under control of a skeletal muscle-specific promoter from mef2c

Genes expressed in skeletal muscle are often required in other tissues. This is particularly the case for cardiac and smooth muscle, both contractile tissues that share numerous characteristics with skeletal muscle, such that targeted inactivation can lead to embryonic lethality prior to a requirement for gene function in skeletal muscle. Thus, it is essential that conditional inactivation approaches are developed to disrupt genes specifically in skeletal muscle. In this report, we describe a transgenic mouse that expresses Cre recombinase under the control of a skeletal muscle-specific promoter from the mef2c gene. Cre expression in this transgenic line is completely restricted to skeletal muscle from early in development and is present in all skeletal muscles, including those of epaxial and hypaxial origins and in fast and slow fibers. This early skeletal muscle-specific Cre line will be a useful tool to define the function of genes specifically in skeletal muscle.