Sequential expression of chicken actin genes during myogenesis

Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha-cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha-Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation.     

Structural changes in chromatin during interphase

The structural organization of the dense chromatin in G₁, S and G₂ of onion meristem cells has been evaluated. A naturally synchronous subpopulation of caffeineinduced binucleate cells was employed. — Fibre size is positively correlated with cell stage in interphase. Fibre size distribution is unimodal in G₁ nuclei with the peak at 12.5 nm in diameter, while in G₂ the distribution is bimodal due to a new population of thicker fibres (22.5 nm in diameter). Separation between fibre centres takes place between mid G₁ and mid S. — Using stereological principles, the length of chromatin fibres integrated in the chromatin patches could be estimated. This length remains constant from mid G₁ to mid S, when a 1.5 fold increase in total DNA takes place. On the other hand, 40 mm/hour of chromatin fibre is integrated in the chromatin patches between mid S and mid G₂. Comparable data of the relative proportion of the different nuclear components have been obtained in each interphase period. — The reported changes provide evidence of a cyclic pattern of chromatin condensation, which may be the structural support for a model of chromatin function in these cycling cells.     

Structural alterations of acidic proteins by acid treatment of chromatin

Summary Recent studies into the properties and biological function of the acidic (non-histone) chromatin proteins have utilized inorganic or organic acids to first remove the histones prior to analysis of the acidic proteins. Examination of the effects of the acid treatment on the DNA and acidic proteins by immunochemistry, circular dichroism, and the ability of the DNA to serve as a template in thein vitro DNA-dependent RNA synthesis, has demonstrated a marked structural change (denaturation) in the proteins and DNA after the acid treatment. Other methods of removing histones, e.g., by high salt or salt and urea, are recommended for studies, especially for those of the biological functions, of the DNA and acidic proteins.     

Structural alterations of chromatin in phase III WI38 human diploid fibroblasts

Circular dichroism spectra of chromatin from phase II (middle-aged) and phase III (old) WI38 human diploid fibroblasts are different. These differences were evident in populations of confluent WI38 cells and were associated with different kinetics when the confluent monolayers were stimulated to proliferate by a nutritional change. The kinetic differences consisted of an increased length of the prereplicative phase and a decreased percentage of cells entering DNA synthesis in the older cell population. The structural differences between the chromatins of middle- and late-passage cells were abolished when both chromatins were extracted with 0.25 M NaCl. Finally, analysis of the 0.25 M NaCl extract showed differences in the gel electrophoretic profiles of the extracted proteins.     

Structural alterations of marrow during inflammation

In response to infections and inflammations, bone marrow reacts to mobilize its granulocyte reserve. Three sets of factors are involved in this mobilization. The structure of the sinus wall is altered and adventitial cells retract to permit interaction of migrating cells with the endothelium. During the maturation process, granulocytes lose their binding potential to the supporting stroma, but their motility, chemotactic ability, and deformability increase. Consequently, they move toward the sinus endothelium with which they interact to enter the circulation. Soluble factors are also involved in granulocyte mobilization. The best characterized of these factors is C3e, an acidic fragment of the alpha chain of C3 with MW of 10-12 KD and ability to bind to granulocyte membrane. Other soluble factors may also be involved, but due to lack of adequate methodology, this area has been relatively underexplored.     

Modulation of histone H3 variant synthesis during the myoblast-myotube transition of chicken myogenesis

We have previously reported that nucleosomal histones are synthesized by cultured, postmitotic myotube cells at 9-29% of the rate in their dividing myoblast precursors (A. M. Wunsch, A. L. Haas, and J. Lough, 1987, Dev. Biol. 119, 85-93). In that study, histones were separated by two-dimensional polyacrylamide gels containing 8 M urea in the first-dimension to optimally separate variants of the H2A class. To separate and compare synthesis of variants in the H2B and H3 classes during myogenesis, 5.75 M urea has been used in the first dimension. Although no changes in the H2B variant pattern were discerned, a dramatic change in H3 variant synthesis was detected, in which a predominance of H3.2 synthesis in dividing myoblasts was almost completely replaced by a lower level of H3.3 synthesis after myotube formation. With increasing differentiation, H3.2 synthesis became undetectable, while H3.3 synthesis continued. Control experiments indicated that these results were not mediated by replicating cells in the myotube cultures, the effects of cytosine arabinoside, or contaminating non-histone proteins. These results suggest that histone H3.2 is replaced by histone H3.3 in nucleosomes during skeletal muscle maturation.     

Specificity of chicken and mammalian transferrins in myogenesis

Chicken transferrins isolated from eggs, embryo extract, serum or ischiatic-peroneal nerves are able to stimulate incorporation of [3H]thymidine, and promote myogenesis by primary chicken muscle cells in vitro. Mammalian transferrins (bovine, rat, mouse, horse, rabbit, and human) do not promote [3H]thymidine incorporation or myotube development. Comparison of the peptide fragments obtained after chemical or limited proteolytic cleavage demonstrates that the four chicken transferrins are all indistinguishable, but they differ considerably from the mammalian transferrins. The structural differences between chicken and mammalian transferrins probably account for the inability of mammalian transferrins to act as mitogens for, and to support myogenesis of, primary chicken muscle cells.     

Differential expression and distribution of chicken skeletal- and smooth-muscle-type alpha-actinins during myogenesis in culture

Antibodies to chicken fast skeletal muscle (pectoralis) alpha-actinin and to smooth muscle (gizzard) alpha-actinin were absorbed with opposite antigens by affinity chromatography, and four antibody fractions were thus obtained: common antibodies reactive with both pectoralis and gizzard alpha-actinins ([C]anti-P alpha-An and [C]anti-G alpha-An), antibody specifically reactive with pectoralis alpha-actinin ([S]anti-P alpha-An), and antibody specifically reactive with gizzard alpha-actinin ([S]anti-G alpha-An). In indirect immunofluorescence microscopy, (C)anti-P alpha-An, (S)anti-P alpha-An, and (C)anti-G alpha- An stained Z bands of skeletal muscle myofibrils, whereas (S)anti-G alpha-An did not. Although (S)anti-G alpha-An and two common antibodies stained smooth muscle cells, (S)anti-P alpha-An did not. We used (S)anti-P alpha-An and (S)anti-G alpha-An for immunofluorescence microscopy to investigate the expression and distribution of skeletal- and smooth-muscle-type alpha-actinins during myogenesis of cultured skeletal muscle cells. Skeletal-muscle-type alpha-actinin was found to be absent from myogenic cells before fusion but present in them after fusion, restricted to Z bodies or Z bands. Smooth-muscle-type alpha- actinin was present diffusely in the cytoplasm and on membrane- associated structures of mononucleated and fused myoblasts, and then confined to membrane-associated structures of myotubes. Immunoblotting and peptide mapping by limited proteolysis support the above results that skeletal-muscle-type alpha-actinin appears at the onset of fusion and that smooth-muscle-type alpha-actinin persists throughout the myogenesis. These results indicate (a) that the timing of expression of skeletal-muscle-type alpha-actinin is under regulation coordination with other major skeletal muscle proteins; (b) that, with respect to expression and distribution, skeletal-muscle-type alpha-actinin is closely related to alpha-actin, whereas smooth-muscle-type alpha- actinin is to gamma- and beta-actins; and (c) that skeletal- and smooth- muscle-type alpha-actinins have complementary distribution and do not co-exist in situ.     

Signaling to the chromatin during skeletal myogenesis: novel targets for pharmacological modulation of gene expression

Cellular differentiation entails an extensive reprogramming of the genome toward the expression of discrete subsets of genes, which establish the tissue-specific phenotype. This program is achieved by epigenetic marks of the chromatin at particular loci, and is regulated by environmental cues, such as soluble factors and cell-to-cell interactions. How the intracellular cascades convert the myriad of external stimuli into the nuclear information necessary to reprogram the genome toward specific responses is a question of biological and medical interest. The elucidation of the signaling converting cues from outside the cells into chromatin modifications at individual promoters holds the promise to unveil the targets for selective pharmacological interventions to modulate gene expression for therapeutic purposes. Enhancing muscle regeneration and preventing muscle breakdown are important goals in the therapy of muscular diseases, cancer-associated cachexia and aging-associated sarcopenia. We will summarize the recent progress of our knowledge of the regulation of gene expression by intracellular cascades elicited by external cues during skeletal myogenesis. And will illustrate the potential importance of targeting the chromatin signaling in regenerative medicine--e.g. to boost muscle regeneration.     

Migration cues induce chromatin alterations

Directed cell migration is a property central to multiple basic biological processes. Here, we show that directed cell migration is associated with global changes in the chromatin fiber. Polarized posttranslational changes in histone H1 along with a transient decrease in H1 mobility were detected in cells facing the scratch in a wound healing assay. In parallel to the changes in H1, the levels of the heterochromatin marker histone H3 lysine 9 tri-methylation were elevated. Interestingly, reduction of the chromatin-binding affinity of H1 altered the cell migration rates. Moreover, migration-associated changes in histone H1 were observed during nuclear motility in the simple multicellular organism Neurospora crassa . Our studies suggest that dynamic reorganization of the chromatin fiber is an early event in the cellular response to migration cues.     

Multiple effects of interferon on myogenesis in chicken myoblast cultures

Effects of chicken interferon on the differentiation of chicken skeletal muscle in vitro were examined. Continuous treatment of chicken myoblast culture with 200 IU/ml of interferon (10 IU/mg protein) resulted in significant inhibition of cell fusion and subsequent myotube formation. However, treatment of myoblast culture with 2 to 200 IU/ml of interferon increased activities of creatine kinase and myokinase in 4- or 6-day cultured muscle cells in a dose-dependent fashion. The effect of interferon on myokinase was less than on creatine kinase. Three-fold increase in creatine kinase activity induced by interferon was not accompanied by the accelerated transition of creatine kinase isozyme from BB- to MM-type. On the other hand, accumulation of acetylcholinesterase in interferon-treated cells at day 6 was suppressed to nearly half the level of control cells. Rates of actin and myosin synthesis in 4-day cultures estimated by pulse-labelling with [35S]methionine were also suppressed to 85% of control cultures. However, a proportion of 35S-labelled actin and myosin in labelled proteins associated with glycerinated cells was not changed by interferon treatment. These results indicate that partially purified interferon has multiple effects on the process of the myogenic differentiation of chicken myoblast in vitro.     

Mitochondrial proliferation during myogenesis

The mitochondrial (mt) DNA content of muscle cells increases about 4-fold during myogenesis. This is apparently the result of continued mtDNA replication after the myoblasts become post-mitotic and nuclear DNA synthesis ceases. The rate of mtDNA synthesis in prefusion cells exceeds that necessary for growth, indicating mt turnover. Eventually, the mtDNA synthesis drops to about one third the prefusion rate, leading to a stable mtDNA content in muscle tissue.     

Fibronectin expression during myogenesis

The biosynthesis and localization of fibronectin during chick muscle differentiation are described. This study employed two monoclonal antibodies, one that selectively killed mononucleated cells and one specific for avian fibronectin. These antibodies allowed precise analyses of fibronectin expression in well-defined cultures of myoblasts or myotubes and avoided the complications of exogenous fibronectin and contamination by fibroblasts or unfused myoblasts. Fibronectin synthesis, as a fraction of total protein synthesis, remains constant at 0.3-0.4% before and after myoblast fusion, suggesting that the absolute rate of fibronectin synthesis may increase somewhat when myotubes synthesize and accumulate myofibrillar proteins. The pattern of fibronectin arrangement does change during myogenesis. In myotube cultures, the appearance of pulse-labeled fibronectin at the cell surface and its secretion into the medium begin after a 2-3-h lag period, in contrast to the 30-min lag period observed in fibroblast cultures. This lag between polypeptide biosynthesis and the exteriorization of the new protein is thus a characteristic of each cell type rather than the protein. All of the major secretory proteins of myogenic cells, including fibronectin and collagenous components, share this 2-3-h intracellular transit time.