The results presented here show that α5-null cells can participate in a wide variety of differentiative processes; animals with a high degree of chimerism in many organs survive and reproduce. The only defect which we observed consistently in chimeric animals containing a significant proportion of α5-null cells was a novel form of muscular dystrophy. This allowed us to focus on the differentiation and survival of muscle cells and tissues and the dependence of these processes on α5β1 integrin.
Earlier results have shown blockade of migration of myoblasts and of their differentiation into myotubes by inhibitory antibodies against various integrin subunits, including β1 (Jaffredo et al., 1988
; Menko et al., 1987), α4 (Rosen et al., 1992
) or α7 (Echtermeyer et al., 1996
; Yao et al., 1996
). However, in vitro differentiation of ES cells and myoblasts lacking β1 or α4 proceeds normally (Yang et al., 1996
; Brakebush et al., 1997) and β1-null and α4-null cells can participate in myogenesis in chimeric mice (Faessler and Meyer, 1995
; Yang et al., 1996
). In the experiments reported here, we obtained similar results for α5-null cells. In vitro, neither α5-null ES cells nor α5-null myoblasts showed any deficit in myogenesis and, in chimeric mice, muscles with a high proportion of α5-null cells could form. These results demonstrate that α5β1 integrin is not essential for the proliferation, migration, or differentiation of myoblasts, myotubes, and skeletal muscles. However, in contrast with the results for α4-null chimeras (Yang et al., 1996
), we observed a significant level of abnormalities in the skeletal muscles of the α5-null chimeras (Figs. –). These muscles showed many characteristics of muscular dystrophy, including giant fibers, central nuclei, vacuoles, fibrosis, and fiber degeneration. Later in life we observed signs of regeneration such as ring fibers. We also observed increased apoptosis and ectopic expression of TN-C (Fig. ) as has been reported for other forms of muscular dystrophy (Settles et al., 1996
These results are reminiscent of the observations of Mayer et al. (1997)
on α7 integrin-deficient mice, which also exhibit muscular dystrophy, and of recent reports of deficiencies in α7β1 in human and murine muscular dystrophies (Hodges et al., 1997
; Mayer et al., 1997
; Vachon et al., 1997). It appears that these two integrins, α5β1 and α7β1, one a receptor for fibronectin and the other a receptor for laminin, are both necessary for long-term integrity of myotubes, although not for their initial development.
α5 is found localized at adhesion plaques (McDonald et al., 1995
) and at the MTJ (Fig. ); α7 is concentrated at the MTJ (McDonald et al., 1995
) where tendons attach. Because these two integrins are present at the points in the fibers where mechanical stress occurs, it suggests an anchoring function of these molecules. Two Drosophila
mutants show a similar situation; in myospheroid
(mutations affecting integrin subunits) muscle differentiation occurs in the absence, respectively, of βPS or αPS2 integrins; however, on contraction, the muscles detach from their attachments (Volk et al., 1990
; Brabant et al., 1993). We can imagine that, in the absence of α7β1 or α5β1, important points of adhesion are lost or weakened and therefore contraction leads to damage to the myotubes. The increase in the number of mitochondria in some fibers of the α5 −/−;+/+ chimeric mice could suggest that, when the fibers cannot function normally, they tend to hypercontract and they need more ATP that requires the formation of a higher number of mitochondria. Other possibilities for the increase in mitochondria include altered differentiation and compensation for the reduced level of α5 integrin.
It is noteworthy that many forms of muscular dystrophy arise from defects in connections to the extracellular matrix. That includes the classical muscular dystrophies arising from defects in dystrophin and its transmembrane linkage via dystroglycans to laminins and in the laminins themselves (Campbell, 1995
). The novel form of muscular dystrophy which we describe here differs from the others, including those caused by α7 integrin deficiencies, in having no obvious connection with laminins. α5β1 has no affinity for laminins and is believed to be specific for fibronectin. The α5-null myoblasts show defects in adherence and survival on fibronectin substrates but behave normally on laminin (Fig. ).
The fact that the muscle defect of the α5-chimeric mice is visible at a very early age (embryonic and postnatal life) and is more attenuated later in life might be due to the high expression, and probable importance, of α5 in embryonic and postnatal muscle followed by later downregulation (McDonald et al., 1995
). In vitro data show that overexpression of α5β1 in myoblasts promotes proliferation and inhibits differentiation, suggesting a proliferative function of this integrin (Sastry et al., 1996
). It is possible that, in the chimeras, α5 is particularly important when a high rate of proliferation is occurring. However, since our mice are chimeras and the muscle fiber is a fusion of α5-null and wild-type cells we cannot exclude the possibility that the defect is partially rescued by the presence of wild-type cells. Another possible reason for amelioration of the phenotype in later life could be gradual replacement of α5-null cells by wild type during regeneration. Consistent with this possibility is the appearance of ring fibers in the older muscles, indicating some fiber regeneration.
We favor the hypothesis that the dystrophy arises from defects in the myofibers themselves, as discussed above. In particular, the time of onset during fetal life corresponds with the period when α5β1 is known to be strongly expressed in muscle cells and the parallels with the muscle defects seen in Drosophila
integrin mutants are suggestive. However, we cannot rule out the possibility that defects or deficits in other α5-null cells, such as interstitial fibroblasts, neurons or Schwann cells or vascular endothelial cells, could contribute to the phenotype observed. We do know that α5-null fibroblasts can assemble FN matrix and migrate and adhere normally (Yang and Hynes, 1996
) and proliferate normally in vitro (Goh, K.L., and R.O Hynes, unpublished data) which argues against a causal defect in fibroblasts without eliminating that possibility.
The reasons for the degenerative changes observed in the muscles deficient in α5β1 remain unclear, as indeed is the case for other muscular dystrophies. Several possible explanations can be imagined. As mentioned above, if α5β1 (and α7β1) are important for maintaining mechanical connections between the myotubes and adjacent structures (e.g., tendons), disruption of the weakened linkage under contraction is a likely initiating cause. Perhaps less likely in this case is a general weakening of the cell surface structure comprising submembranous cytoskeleton connected to the basal lamina. Another possibility is that the apoptosis (Fig. ) could be a causative event rather than a secondary consequence. Precedent exists for cells' being dependent on specific integrin-matrix adherence for cell survival (Zhang et al., 1995
) and such dependences include dependence on α5β1–fibronectin interactions (Zhang et al., 1995
). However, our in vitro data somewhat argue against this idea without ruling it out. The α5-null myoblasts do indeed show increased apoptosis when plated on fibronectin but do not when plated on laminin (Fig. ). Myotubes are surrounded by a basal lamina rich in laminin, although also containing fibronectin. Thus, although it is possible that adherence to fibronectin via α5β1 is specifically necessary for myotube survival, it seems more likely that any such requirement for attachment to basal lamina is satisfied by connection to laminin via α7β1 or via dystroglycans. Whatever the detailed cause–effect relationships leading to fiber degeneration and muscular defects in the mice, the results reported here reveal a novel form of muscular dystrophy.