S. macrurus myogenic regulatory factors (MRFs) induce mammalian skeletal muscle differentiation; evidence for functional conservation of MRFs

Abstract

The current-producing cells of the electric organ (EO), i.e., electrocytes, in Sternopygus macrurus derive from skeletal muscle fibers. Mature electrocytes are not contractile but they do retain some muscle proteins, are multinucleated, and receive cholinergic innervation. Electrocytes express the myogenic regulatory factors (MRFs) MyoD, myogenin, Myf5 and MRF4 despite their incomplete muscle phenotype. Although S. macrurus MRFs share functional domains that are highly conserved and their expression is confined to the myogenic lineage, their capability to induce the muscle phenotype has not been determined. To test the functional conservation of S. macrurus MRFs to transcriptionally activate skeletal muscle gene expression and induce the myogenic program, we transiently over-expressed S. macrurus MyoD (SmMyoD) and myogenin (SmMyoG) in mouse C3H/ 10T1/2 and NIH3T3 embryonic cells. RT-PCR and immunolabeling studies showed that SmMyoD and SmMyoG efficiently can convert these two cell lines into multinucleated myotubes that expressed differentiated muscle markers. The levels of myogenic induction by SmMyoD and SmMyoG were comparable to those obtained with mouse MRF homologs. Furthermore, SmMyoD and SmMyoG proteins were able to induce mouse MyoD and myogenin in C3H/10T1/2 cells. We conclude that S. macrurus MRFs are functionally conserved as they can transcriptionally activate skeletal muscle gene expression and induce the myogenic program in mammalian non-muscle cells. Hence, these data suggest that the partial muscle phenotype of electrocytes is not likely due to differences in the MRF-dependent transcriptional program between skeletal muscle and electric organ.

Differential expression of genes and proteins between electric organ and skeletal muscle in the mormyrid electric fishBrienomyrus brachyistius

“…assumptions regarding the identities of specific genes that might be differentially expressed (Patterson and Zakon, 1996;Patterson and Zakon, 1997;Cuellar et al, 2006;Kim et al, 2008;Kim et al, 2009;Kim et al, 2004;Unguez and Zakon, 1998a;Unguez and Zakon, 1998b;Zakon and Unguez, 1999). Unlike these prior approaches, our use of the SSH technique allows for the construction of a dataset for which fewer assumptions have been made.…”

“…Differential expression of these transcription factors may therefore provide an attractive hypothesis to explain physiological and morphological differences between EOs and skeletal muscle. A series of studies (Kim et al, 2004: Kim et al, 2008Kim et al, 2009) examined the transcriptional patterns and roles of several MRFs and their co-factors including MyoD, Myogenin, MYF5, MRF4, MEF2C, ID1 and ID2 in the gymnotiform Sternopygus macurus. These studies found little difference in expression levels between SM and EO, and that several of the myogenic regulatory factors cloned from S. macurus retain their ability to induce normal muscle development in mouse cultured myoblasts (Kim et al, 2008;Kim et al, 2009).…”

“…Considered in the light of previous denervation studies (Szabo and Kirschbaum, 1983), these findings raise the possibility that EO development in mormyrids may not be initiated through the same developmental processes. This is especially intriguing given that the gymnotiform EO can be regenerated upon injury (Cuellar et al, 2006;Kim et al, 2008;Kim et al, 2009;Kim et al, 2004;Unguez and Zakon, 1998a;Unguez and Zakon, 1998b;Zakon and Unguez, 1999;Patterson and Zakon, 1996;Patterson and Zakon, 1997), whereas the mormyrid EO cannot.…”

“…Several studies over the past two decades have contributed to our understanding of gene expression and development in gymnotiform EOs, in particular with regard to the role of innervation (Cuellar et al, 2006;Kim et al, 2008;Kim et al, 2009;Kim et al, 2004;Unguez and Zakon, 1998a;Unguez and Zakon, 1998b;Zakon and Unguez, 1999;Patterson and Zakon, 1996;Patterson and Zakon, 1997). Comparative studies between gymnotiforms and mormyrids, however, are presently limited because no studies have described gene expression in the mormyrid EO, which is the focus of the present study.…”

Mechanisms of muscle gene regulation in the electric organ ofSternopygus macrurus

“…Expression analyses revealed that mature electrocytes transcribe all four MRF genes, with expression levels of myogenin, MRF4 and Myf5 transcripts being significantly higher than those detected in skeletal muscle fibers ( Fig.3A) (Kim et al, 2004;Kim et al, 2008). Immunolabeling studies using S. macrurus-specific antibodies against MyoD and myogenin confirmed that these MRFs are translated and localized in nuclei of muscle fibers and electrocytes ( Fig.3B) (Kim et al, 2009). Together, these data reveal that expression of multiple MRFs is not sufficient to induce the non-contractile electrocytes to fully express the striated muscle program.…”