Specification and differentiation by the myogenic bHLH proteins
As we have seen, muscle cells come from two cell lineages in the somite. In both instances, paracrine factors instruct the myotome cells to become muscles by inducing them to synthesize the MyoD protein (Maroto et al. 1997; Tajbakhsh et al. 1997). In the lateral portion of the somite, which forms the hypaxial muscles, factors from the surrounding environment induce the Pax3 transcription factor. In the absence of other inhibitory transcription factors (such as those found in the sclerotome cells), Pax3 then activates the genes encoding two muscle-specific transcription factors, Myf5 and MyoD. In the medial region of the somite, which forms the epaxial muscles, MyoD is induced through a slightly different pathway. MyoD and Myf5 belong to a family of transcription factors called the myogenic bHLH (basic helix-loop-helix) proteins (sometimes also referred to as the MyoD family). The proteins of this family all bind to similar sites on the DNA and activate muscle-specific genes. For instance, the MyoD protein appears to directly activate the muscle-specific creatine phosphokinase gene by binding to the DNA immediately upstream from it (Lassar et al. 1989), and there are two MyoD-binding sites on the DNA adjacent to the genes encoding a subunit of the chicken muscle acetylcholine receptor (Piette et al. 1990). MyoD also directly activates its own gene. Therefore, once the myoD gene is activated, its protein product binds to the DNA immediately upstream of the myoD gene and keeps this gene active.
While Pax3 is found in several other cell types, the myogenic bHLH proteins are specific for muscle cells. Any cell making a myogenic bHLH transcription factor such as MyoD or Myf5 is committed to becoming a muscle cell. Transfection of genes encoding any of these myogenic proteins into a wide range of cultured cells converts those cells into muscles (Thayer et al. 1989; Weintraub et al. 1989).
WEBSITE
14.3 Myogenic bHLH proteins and their regulators. Since the MyoD protein and its relatives are so powerful that they can turn nearly any cell into a muscle cell, the synthesis of this protein has to be inhibited at numerous steps. Numerous inhibitors of MyoD family gene expression and protein function have been found. http://www.devbio.com/chap14/link1403.shtml
Muscle cell fusion
The myotome cells producing the myogenic bHLH proteins are the myoblasts—committed muscle cell precursors. Experiments with chimeric mice and cultured myoblasts showed conclusively that these cells align together and fuse to form the multinucleated myotubes characteristic of muscle tissue. Thus, the multinucleated myotube cells are the product of several myoblasts joining together and dissolving the cell membranes between themselves (Konigsberg 1963; Mintz and Baker 1967).
Muscle cell fusion begins when the myoblasts leave the cell cycle. As long as particular growth factors (particularly fibroblast growth factors) are present, the myoblasts will proliferate without differentiating. When these factors are depleted, the myoblasts stop dividing, secrete fibronectin onto their extracellular matrix, and bind to it through α5β1 integrin, their major fibronectin receptor (Menko and Boettiger 1987; Boettiger et al. 1995). If this adhesion is experimentally blocked, no further muscle development ensues, so it appears that the signal from the integrin-fibronectin attachment is critical for instructing the myoblasts to differentiate into muscle cells (Figure 14.10).
Figure 14.10
Conversion of myoblasts into muscles in culture. (A) Determination of myotome cells by paracrine factors. (B) Committed myoblasts divide in the presence of growth factors (primarily FGFs), but show no obvious muscle-specific proteins. (C-D) When the growth (more...)
The second step is the alignment of the myoblasts together into chains. This step is mediated by cell membrane glycoproteins, including several cadherins and CAMs (Knudsen 1985: Knudsen et al. 1990). Recognition and alignment between cells takes place only if the two cells are myoblasts. Fusion can occur even between chick and rat myoblasts in culture (Yaffe and Feldman 1965); the identity of the species is not critical.
The third step is the cell fusion event itself. As in most membrane fusions, calcium ions are critical, and fusion can be activated by calcium ionophores, such as A23187, that carry calcium ions across cell membranes (Shainberg et al. 1969; David et al. 1981). Fusion appears to be mediated by a set of metalloproteinases called meltrins. These proteins were discovered during a search for myoblast proteins that would be hom*ologous to fertilin, a protein implicated in sperm-egg membrane fusion. Yagami-Hiromasa and colleagues (1995) found that one of these meltrins (meltrin-α) is expressed in myoblasts at about the same time that fusion begins, and that antisense RNA to the meltrin-α message inhibited fusion when added to myoblasts.
WEBSITE
14.4 Myotube formation. Multinucleated myotubes could arise either as (1) a fusion event between several mononuclear myoblasts, or (2) as a string of mitoses within a single myoblast. The former is thought to be the mechanism for myotube formation in skeletal muscle; the latter is thought to be the way heart myotubes are formed. http://www.devbio.com/chap14/link1404.shtml
