Cellular Localization of Muscle and Nonmuscle Actin mRNAs in Chicken Primary Myogenic Cultures: The Induction of a-Skeletal Actin mRNA Is Regulated Independently of t -Cardiac Actin Gene Expression

Specific DNA fragments complementary to the 3' untranslated regions of the 13-, ~t-cardiac, and ~t-skeletal actin mRNAs were used as in situ hybridization probes to examine differential expression and distribution of these mRNAs in primary myogenic cultures. We demonstrated that prefusion bipolar-shaped cells derived from day 3 dissociated embryonic somites were equivalent to myoblasts derived from embryonic day 11-12 pectoral tissue with respect to the expression of the ~t-cardiac actin gene. Fibroblasts present in primary muscle cultures were not labeled by the ~t-cardiac actin gene probe. Since virtually all of the bipolar cells express a-cardiac actin mRNA before fusion, we suggest that the bipolar phenotype may distinguish a committed myogenic cell type. In contrast, a-skeletal actin mRNA accumulates only in multinucleated myotubes and appears to be regulated independently from the ct-cardiac actin gene. Accumulation of a-skeletal but not ~t-cardiac actin mRNA can be blocked by growth in Ca2+-deficient medium which arrests myoblast fusion. Thus, the sequential appearance of a-cardiac and then ~t-skeletal actin mRNA may result from factors that arise during terminal differentiation. Finally, the 13-actin mRNA was located in both fibroblasts and myoblasts but diminished in content during myoblast fusion and was absent from differentiated myotubes. It appears that in primary myogenic cultures, an asynchronous stage-dependent induction of two different a-striated actin mRNA species occurs concomitant with the deinduction of the nonmuscle fi-actin gene. M VOGENESIS in culture parallels embryonic muscle development to the extent that myoblasts proliferate, withdraw from the cell cycle, fuse to form myotubes, and elaborate functional myofibrillar sarcomeres (reviewed in reference 30). Biochemical differentiation, as evidenced by the induction of muscle-specific gene products and the repression of subsets of nonmuscle genes, normally occurs after myoblast terminal commitment and fusion. Myoblasts grown in Ca2÷-deficient medium have demonstrated that biochemical differentiation does not require irreversible withdrawal from the cell cycle or the formation of multinucleated myotubes (12, 23, 25, 31, 33). Myoblast fusion may also be inhibited by certain N-carbobenzyloxy dipeptide amides that compete with the protein substrate of a soluble cytoplasmic metalloendoprotease (MEPr) t (9, 10). Recent evidence suggests that MEPrs may be required for biochemical differentiation (3, 12) as well as fusion. We examined the switch in expression of the chicken actin 1. Abbreviations used in this paper: LCM, low Ca2+-containing medium; MEPr, metalloendoprotease. multigene family during the terminal differentiation of skeletal myoblasts (29). We recently showed that primary cultures of chicken myoblasts express specific actin mRNAs in a sequential pattern leading to terminal differentiation (17). Nonmuscle 13-actin mRNA was detected predominantly in proliferating myogenic cultures and declines in content as myogenesis continues. Within 24 h after the plating of dissociated embryonic myoblasts, a-cardiac actin mRNA is induced and later is maintained as the major postfusion actin mRNA. In contrast, a-skeletal actin mRNA, the predominant skeletal muscle actin mRNA in vivo, is induced only after fusion is under way (17). We reasoned that the temporal pattern of induction and deinduction of actin genes could be explained by the spatial distribution of actin mRNAs among cells at different stages of myogenesis. Therefore, several questions regarding myogenic regulation of the actin multigene family remained unanswered by previous studies. Specifically: (a) Does the low, but significant, level of 13-actin mRNA in late muscle cultures result from low constitutive expression in myotubes or from abundant 13-actin mRNA in the relatively few residual © The Rockefeller University Press, 0021-9525/88/06/2077/10 $2.00 The Journal of Cell Biology, Volume 106, June 1988 2077-2086 2077 on July 8, 2017 jcb.rress.org D ow nladed fom