Developmental regulation of the Drosophila tropomyosin I (TmI) gene is controlled by a muscle activator enhancer region that contains multiple cis-elements and binding sites for multiple proteins

Developmental gene regulation in vertebrate somatic muscles involves the cooperative interaction of MEF2 (myocyte-specific enhancer-binding factor 2) and members of the b-HLH (basic helix-loop-helix) family of myogenic factors. Until recently, however, nothing was known about the factors that control the developmental regulation of muscle genes during embryogenesis in Drosophila. The Drosophila Tropomyosin I (TmI) gene contains a proximal and distal muscle enhancer within the first intron that regulates its expression in embryonic/larval and adult muscles. We have recently shown that the 355-bp proximal enhancer contains a binding site for the Drosophila homologue of vertebrate MEF2 and that MEF2 acts cooperatively with a basal level muscle activator region to direct high level muscle expression in transgenic flies. The 92-bp muscle activator region, however, does not contain any consensus E-box (CANNTG) binding site sequences for b-HLH myogenic factors, suggesting the MEF2 may interact with other factors to regulate muscle genes in Drosophila. In this study we have used mutation analysis and germ-line transformation to analyze the cis-acting elements within the muscle activator region that regulate its expression in transgenic flies. We have identified a 71-bp region that is sufficient for low basal level temporal- and muscle-specific expression in the embryo, larva, and adult. Substitution mutations within the muscle activator region have identified several cis-element regions spanning 60-bp that are required for either full or partial muscle activator function. An analysis of proteins that bind to this region by gel mobility shift assay and copper nuclease footprinting has allowed us to identify the sites in this region at which multiple proteins complex and interact. We propose that these cis-elements and the proteins that they bind regulate muscle activator function and together with MEF2 are capable of regulating high level muscle expression. Dev. Genet. 20:297–306, 1997. © 1997 Wiley-Liss, Inc.     

Control of morphogenetic cell movements in the early zebrafish myotome

As the vertebrate myotome is generated, myogenic precursor cells undergo extensive and coordinated movements as they differentiate into properly positioned embryonic muscle fibers. In the zebrafish, the "adaxial" cells adjacent to the notochord are the first muscle precursors to be specified. After initially differentiating into slow-twitch myosin-expressing muscle fibers, these cells have been shown to undergo a remarkable radial migration through the lateral somite, to populate the superficial layer of slow-twitch muscle of the mature myotome. Here we characterize an earlier set of adaxial cell behaviors; the transition from a roughly 4x5 array of cuboidal cells to a 1x20 stack of elongated cells, prior to the migration event. We find that adaxial cells display a highly stereotypical series of behaviors as they undergo this rearrangement. Furthermore, we show that the actin regulatory molecule, Cap1, is specifically expressed in adaxial cells and is required for the progression of these behaviors. The requirement of Cap1 for a cellular apical constriction step is reminiscent of similar requirements of Cap during apical constriction in Drosophila development, suggesting a conservation of gene function for a cell biological event critical to many developmental processes.     

In vitro myogenic and adipogenic differentiation model of genetically engineered bovine embryonic fibroblast cell lines

Our current understanding of muscle and adipose tissue development has been largely restricted to the study of murine myogenic and adipogenic cell lines, since attempts to establish these cell lines from other species have met with only limited success. Here we report that a spontaneously immortalized bovine embryonic fibroblast cell line (BEFS) undergoes differentiation into adipogenic or myogenic lineages when ectopically transduced with PPARγ2 (an adipogenic lineage determinant) or MyoD (a myogenic lineage determinant) and grown in adipogenic and myogenic differentiation culture media (ADCM and MDCM, respectively). We also found that PPARγ2-overexpressing BEFS cells (BEFS-PPARγ2) grown in ADCM with or without the PPARγ2 ligand, troglitazone, preferentially differentiate into adipogenic cells in the presence of ectopic MyoD expression. Ectopic expression of PPARγ2 in the inducible MyoD-overepxressing BEFS cells (BEFS-TetOn-MyoD) completely suppresses myogenic differentiation and leads to a significant increase in adipogenic differentiation, suggesting that the adipogenic differentiation program might be dominant. Therefore, BEFS, BEFS-PPARγ2, and BEFS-TetOn-MyoD would be a valuable biological model for understanding a fundamental principle underlying myogenic and adipogenic development, and for isolating various genetic and chemical factors that enable muscle and adipocyte differentiation.     

Sonic hedgehog inhibits the terminal differentiation of limb myoblasts committed to the slow muscle lineage

The proliferation, differentiation, and fusion of a small number of myogenic precursor cells must be precisely regulated during development to ensure the proper size, organization, and function of the limb musculature. We have examined the role of Sonic hedgehog (Shh) in these processes by both augmentation and inhibition of the Shh-mediated signaling pathway. Our data show that Shh regulates muscle development by repressing the terminal differentiation of early myogenic precursor cells and does not function as a myoblast mitogen. Shh function in hypaxial muscle appears to be spatially restricted to the early myoblast population within the ventral muscles of the posterior region of the limb. Furthermore, Shh appears to act as a permissive, rather than an inductive, signal for slow MyHC expression in myoblasts. Our data thus provide the foundation for a new hypothesis for Shh function in hypaxial skeletal muscle development.     

Regulation of myogenic stem cell behaviour by vessel cells: The "ménage à trois" of satellite cells,periendothelial cells and endothelial cells

In skeletal muscle, satellite cells, that are responsible of muscle repair, are localized close to capillaries. Although angiogenesis is known for a long time to be crucial for muscle repair and satellite cell survival, cellular interplays between vessel cells and satellite/myogenic cells have been poorly explored. We analyzed the interrelationships between myogenic cells, endothelial cells, and periendothelial cells that includes smooth muscle cells and endomysial fibroblasts. We found that endothelial cells strongly stimulate myogenic cell growth and, inversely, myogenic cells increase angiogenesis. VEGF plays a essential role in this bidirectional interaction. On the contrary, periendothelial cells promote the return to quiescence of a subset of muscle precursor cells to quiescence that ensures self-renewal of adult muscle stem cells. We have shown that Angiopoietin-1/Tie-2 signalling controls the entry into quiescence. We propose that during muscle regeneration, i.e. while vessels are not stabilized, endothelial cells and myogenic cells interact with each other to promote both myogenesis and angiogenesis, that have been shown to be concomitant processes in several models. On the other hand, once homeostasis of muscle is reached, the proximity of satellite cells and periendothelial cells allows the responsiveness of satellite cells, that bear Tie-2 receptor, to the secretion of Angiopoietin-1 by periendothelial cells, that, in the same time, stabilize vessels by promoting quiescence of endothelial cells.