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Apoptotic myonuclei appear during myogenesis and in diseased muscles. To investigate cell death regulation in skeletal muscle, we examined how members of the Bcl-2 family of apoptosis regulators are expressed and function in the C2C12 muscle cell line and in primary muscle cells at different stages of development. Both anti-apoptotic (Bcl-W, Bcl-X(L)) and pro-apoptotic (Bad, Bak, Bax) members of the Bcl-2 family were expressed in developing skeletal muscle in vivo. Each was also expressed in embryonic (E11-12), fetal (E15-16), and neonatal muscle stem cells, myoblasts, and myotubes in vitro. In contrast, Bcl-2 expression was limited to a small group of mononucleate, desmin-positive, myogenin-negative muscle cells that were seen in fetal and neonatal, but not embryonic, muscle cell cultures. The cell surface protein Sca-1, which is associated with muscle and blood stem cells, was found on approximately 1/2 of these Bcl-2-positive cells. Loss of Bcl-2 did not affect expression of other family members, because neonatal muscles of wild-type and Bcl-2-null mice had similar amounts of Bcl-X(L), Bcl-W, Bad, Bak, and Bax mRNAs. Loss of Bcl-2 did have functional consequences; however, because neonatal muscles of Bcl-2-null mice had only approximately 2/3 as many fast muscle fibers as muscles in wild-type mice. Thus, Bcl-2 function is required for particular stages of fetal and postnatal myogenesis., (Copyright 2001 Wiley-Liss, Inc.)

We show that Bcl-2 expression in skeletal muscle cells identifies an early stage of the myogenic pathway, inhibits apoptosis, and promotes clonal expansion. Bcl-2 expression was limited to a small proportion of the mononucleate cells in muscle cell cultures, ranging from approximately 1-4% of neonatal and adult mouse muscle cells to approximately 5-15% of the cells from the C2C12 muscle cell line. In rapidly growing cultures, some of the Bcl-2-positive cells coexpressed markers of early stages of myogenesis, including desmin, MyoD, and Myf-5. In contrast, Bcl-2 was not expressed in multinucleate myotubes or in those mononucleate myoblasts that expressed markers of middle or late stages of myogenesis, such as myogenin, muscle regulatory factor 4 (MRF4), and myosin. The small subset of Bcl-2-positive C2C12 cells appeared to resist staurosporine-induced apoptosis. Furthermore, though myogenic cells from genetically Bcl-2-null mice formed myotubes normally, the muscle colonies produced by cloned Bcl-2-null cells contained only about half as many cells as the colonies produced by cells from wild-type mice. This result suggests that, during clonal expansion from a muscle progenitor cell, the number of progeny obtained is greater when Bcl-2 is expressed.

We show that members of the POU homeodomain family are among the transcription factors expressed in developing mouse skeletal muscle. From a cDNA library prepared from fetal muscle mRNA, we cloned a cDNA identical to that of Brn-4, a POU class II gene previously cloned from neural tissues. In limb muscle, we found that Brn-4 mRNA expression was highest at embryonic days 15-18, declined-after birth, and was undetectable in adults. The mRNAs of two additional POU genes, Emb (POU class VI) and Oct-1 (POU class II), were also expressed in developing muscle and, unlike Brn-4, continued to be expressed in postnatal and adult muscles. In skeletal muscle, expression of Brn-4 is myogenin-dependent, because muscles from myogenin-deficient fetuses contained much less Brn-4 mRNA than muscles from myogenin-expressing littermates. In contrast, expression of Emb was the same in the presence or absence of myogenin. The distinct pattern of Brn-4 mRNA expression and its dependence on a myogenic regulatory factor suggest that Brn-4 is part of the network of interacting transcription factors that control muscle-specific gene expression during mammalian myogenesis.