The paired box transcription factor Pax7 was isolated by representational difference analysis as a gene specifically expressed in cultured satellite cell-derived myoblasts. In situ hybridization revealed that Pax7 was also expressed in satellite cells residing in adult muscle. Cell culture and electron microscopic analysis revealed a complete absence of satellite cells in Pax7(-/-) skeletal muscle. Surprisingly, fluorescence-activated cell sorting analysis indicated that the proportion of muscle-derived stem cells was unaffected. Importantly, stem cells from Pax7(-/-) muscle displayed almost a 10-fold increase in their ability to form hematopoietic colonies. These results demonstrate that satellite cells and muscle-derived stem cells represent distinct cell populations. Together these studies suggest that induction of Pax7 in muscle-derived stem cells induces satellite cell specification by restricting alternate developmental programs.

Myotonic dystrophy (DM) is the most common inherited neuromuscular disease in adults, with a global incidence of 1 in 8000 individuals. DM is an autosomal dominant, multisystemic disorder characterized primarily by myotonia and progressive muscle weakness. Genomic and complementary DNA probes that map to a 10-kilobase Eco RI genomic fragment from human chromosome 19q13.3 have been used to detect a variable length polymorphism in individuals with DM. Increases in the size of the allele in patients with DM are now shown to be due to an increased number of trinucleotide CTG repeats in the 3' untranslated region of a DM candidate gene. An increase in the severity of the disease in successive generations (genetic anticipation) is accompanied by an increase in the number of trinucleotide repeats. Nearly all cases of DM (98 percent or 253 of 258 individuals) displayed expansion of the CTG repeat region. These results suggest that DM is primarily caused by mutations that generate an amplification of a specific CTG repeat.

Over the past years, several studies have unraveled important mechanisms by which the four myogenic regulatory factors (MRFs: MyoD, Myf-5, myogenin, and MRF4) control the specification and the differentiation of the muscle lineage. Early experiments led to the hypothesis that these factors were redundant and could functionally replace one another. However, recent experiments using in vivo and in vitro models have demonstrated that in fact different aspects of the myogenic program are controlled by different factors in vivo, suggesting that these factors play distinct roles during myogenesis. The activity of the MRFs during proliferation and differentiation of muscle precursor cells has clearly been demonstrated to be dependent on specific cell-cycle control mechanisms as well as distinct interactions with other regulatory molecules, such as the ubiquitously expressed E proteins and several other transcription factors. Furthermore, the observation that the MRFs can recruit chromatin remodeling proteins has shed some light on the mechanisms by which the MRFs activate gene expression. Recently, a functional role for MyoD during satellite cell activation and muscle repair has been identified in vivo, which cannot be substituted for by the other MRFs. This has put forward the hypothesis that these factors also play specific biological roles following muscle injury and repair.

To gain insight into the regeneration deficit of MyoD−/− muscle, we investigated the growth and differentiation of cultured MyoD−/− myogenic cells. Primary MyoD−/− myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD−/− myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD−/− myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD−/− cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD−/− myogenic cells. Mixing of lacZ-labeled MyoD−/− cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD−/− myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.

We hypothesize that PDZK1 and NHERF-1 establish an extended network beneath the apical membrane to which membrane proteins and regulatory components are anchored.