Donald Gullberg, Teet Velling, Lars Lohikangas, Carl-Fredrik Tiger

Department of Animal Physiology, Uppsala University, BMC, Box 596, S-751 24 Uppsala , Sweden

Received 9/7/98 Accepted 9/21/98

2. IN VIVO myogenesis

2.1. Early vertebrate myogenesis

Vertebrate myogenesis involves a number of steps controlled by a variety of signals and is ultimately orchestrated by regulatory events at the gene level in muscle cells. Unlike many other cell types in the body the muscle fiber is multinuclear and there is evidence that transcriptional activity varies between nuclei within the muscle fiber (1-3).

The current knowledge about the complex inductive extracellular signals as well as the ensuing transcriptional events during myogenesis have been the subject of recent reviews (4-6). we will try and put some of this knowledge into the context of cellular interactions with the extracellular matrix (ECM). During vertebrate myogenesis the paraxial mesoderm becomes segmented into epithelial ball like structures named somites. Somites differentiate into sclerotome and dermomyotome. The dermomyotome will develop further into the dermatome and the myotome. With the exception of some head muscles, skeletal muscles arise from the dermomyotome. Axial muscles are derived from different regions in the myotome whereas appendicular muscles arise from a cell population in the ventrolateral part of the dermomyotome (see figure 1). The molecular cues governing these early steps of myogenensis are currently subject to intense investigation (reviewed in (7, 8). Much of these studies are currently performed in mouse. Axial structures such as the neural tube and the notochord are needed for formation of epaxial muscles (deep back and intercostal muscles) while cues from surface ectoderm and lateral mesoderm are needed for hypaxial (trunk) muscle formation. Soluble molecules that act very early in myogenesis include Wnts, sonic hedgehog and bone morphogenetic protein-4 (reviewed in (6)). The transcription factor pax-3 is needed for the formation of both axial and appendicular muscles, but not for head muscle development (9). Myogenic regulatory factors (MRFs) are transcription factors of the bHLH class and contain the members MyoD, Myf-5, myogenin and MRF-4 (10). Specific MRF members are expressed in different regions of the differentiating myotome. In the dorsal part of the myotome,which gives rise to epaxial muscles, Myf-5 is an early orchestrator of determination events. In the more ventral part MyoD appears to play an important role for hypaxial muscle formation (6, 8, 10). At these early steps a number of cell migration events occur. In Myf-5 defective embryos, myotomal cells migrate and position themselves abnormally (11). Committed myogenic cells in the ventrolateral tip of the somite which do not yet express MRFs express the c-Met receptor during the migration into the limbs (12). c-Met is a tyrosine kinase receptor for scatter factor. Recent in vitro data have indicated that in myoblasts an autocrine loop is active for scatter factor (13) and that the c-Met receptor signals via Grb-2 in the myogenic lineage (14). Whether an autocrine loop is also operative during myogenic migration at this step in vivo is not known. When the somite-derived cells have stopped migrating they start expressing MRFs and now can be identified as myoblasts. Axial and appendicular myoblast that undergo the first stage of differentiation are said to undergo primary myogenesis (15). Using the primary myotubes as a scaffold, a distinct myoblasts population-

the secondary myoblasts - line up under the basement membrane of primary myotubes, fuse with each other and form secondary myotubes (see figure 1). Subsequent to these early events a number of important reorganisation events need to take place in order to achieve the final muscle pattern. Secondary muscle fibers form an independent basement membrane. Muscles split, become innervated, achieve their final pattern and grow. At the early times of muscle development the myotube end points are thought to be important for muscle splitting and muscle growth (1, 16). Adult muscle fibers have an elaborate system for force transmission, mainly via the specialized endpoints at attachments to tendons, called myotendinous junctions.

Figure 1. Schematic representation of integrin expression during different steps of myogenesis in mouse. Schematic representation of myogenesis: Skeletal muscle originate from two regions in the differentiating somite. Axial muscle form from the myotome, while appendicular muscle arise from the ventrolateral tip of the dermomyotome. At the level of the forelimb and hindlimb cells migrate out to form limb muscle. In the schematic representation of muscle formation only maturation of appendicular muscle is shown. Except for the type of cell migration observed for limb muscle, axial muscle form in similar steps. Integrin expression: Data on integrin expression during mouse muscle development has been summarized. At the somite stage, staining usually refers to myotome staining, i.e staining in the cells destined to become axial muscle.

Unlike cardiac muscle, skeletal muscle has the capacity to regenerate in response to injury. The basis for this capacity is laid down during fetal development by a distinct cell population called satellite cells. This population of myoblasts will normally not differentiate. In the case of muscle injury, part of these cells will remain as stem cells whereas others will differentiate into new muscle fibers. Recently FGF-6 and MyoD have been shown to be important for proper satellite cell activation in response to injury (17, 18). Events during myogenesis suggested to depend on cell-ECM interactions include: somite formation, cell determination events, early cell migratory events, myogenic differentiation, basement membrane assembly, muscle positioning, myotube alignment, muscle splitting, innervation and, muscle fiber stability. With modern molecular biology tools, essentially all these events can be studied in vivo.

2.2. Lessons from Drosophila myogenesis

Drosophila has served as a useful model for studies of several complex developmental processes. No endoskeleton exists in invertebrates and the somatic musculature is used for larval crawling and later in adult insect jumping and flight. Despite the different anatomical considerations the conservation of basic molecular mechanisms during muscle formation is striking (19, 20). Recently several mutations in Drosophila have shed light on the complexity of the process of muscle formation (19, 21, 22). A number of mutations inactivating transcription factors, signaling molecules and cell membrane proteins, all affect muscle formation. For some of the identified proteins, corresponding orthologues have not yet been found in vertebrates. For others orthologues exist, but their involvement in vertebrate myogenesis is not clear. For those inductive mechanisms where the pathways start to pan out the emerging pattern suggest that conserved genes are used in different ways in invertebrates and vertebrates. As pointed out by Baylies et al (19) this might prompt us to look at vertebrate myogenesis with "new eyes" - maybe things are not what they appear to be. Drosophila will continue to be a valuable system to study fundamental molecular aspects of myogenesis.