| Dab2 is an intracellular adaptor protein and a tumor suppressor. In mouse embryos, Dab2 was found to be expressed in the dermomyotome of somites from E8.5 to E11.0 using immunofluorescence staining, with expression first detected in the medial aspect of the dermomyotome at E8.5 and then co-localized with the early muscle markers Pax3 and Myf5 at the ventrolateral lip of the dermomyotome at E10.5. From E11.5 to E14.5, Dab2 was expressed in muscle masses of limb buds and the trunk. Dab2 expression in skeletal muscles was gradually decreased after birth. These observations suggested potential roles of Dab2 in the skeletal muscle myogenesis. In addition, since the normal development of skeletal muscles requires proper signal transduction, and Dab2 has been known to be involved in the MAPK, TGF-beta and Wnt signaling pathways, Dab2 may therefore be important for the muscle development.;To determine the role of Dab2 in the skeletal muscle development, Xenopus laevis embryos and C2C12 myoblasts were employed as in vivo and in vitro models, respectively. In situ hybridization results showed that XDab2 was expressed in somites of Xenopus embryos and co-localized with the muscle markers XPax3, XMyoD, XMef2c and XMyos. Knockdown of XDab2 expression with antisense morpholinos down regulated the expression of several muscle markers in somites including XPax3, XMyf5, XMef2c, XMyoS and XAC100. Down-regulation of MHC and 12/101 were also observed in whole mount preparations and transverse sections of XDab2 morpholino-injected embryos after immunohistochemical staining.;The C2C12 cell line derived from mouse muscle satellite cells was then employed as an in vitro model to determine the role of Dab2 during early muscle development. When C2C12 myoblasts were induced to differentiate into myotubes, Dab2 expression was simultaneously increased at RNA and protein levels. Dab2 over-expression after transfection with Dab2 plasmids resulted in enhanced myoblast fusion and increased numbers of myotubes. Conversely, suppression of Dab2 expression with miRNAs resulted in reduced myoblast fusion and decreased numbers of myotubes. Lentiviral shRNA-mediated Dab2 stable knockdown reduced myotube formation in 2 representative stable clones, clone 5-2 and clone 5-7. Western blot analysis showed that expression of phospho-p38 MAPK was down-regulated in clone 5-2 and 5-7. Dab2 re-expression through plasmid-mediated transient transfection in clone 5-2 could partially restore the myotube formation. These observations therefore suggested that Dab2 plays essential roles in the formation of myotubes.;Comprehensive profiling of differentially expressed genes was performed with the Affymetrix microarray analysis between the Dab2-knockdown clone 5-2 and the C2C12 parental cell line. As compared to the parental cells, the clone 5-2 showed significant changes in the expression of 235 probe sets representing 155 genes (p<0.05) with 2 folds or greater changes. Among the 155 genes, 127 were down-regulated, while 28 up-regulated. qRT-PCR results were found to be consistent with the microarray results. Functions of the differentially expressed genes were found to be significantly associated with the development and functions of the muscular system. Knockdown of Dab2 affected the genes involved in muscle contraction, the contraction of striated muscle, differentiation of muscle precursor cells, and the development of skeletal muscle fibers. A network analysis and a gene expression study revealed that Mef2c down-regulation was related to the inhibition of myogenic differentiation in the clone 5-2. Furthermore, forced expression of Mef2c in the clone 5-2 could rescue the myogenic differentiation.;In conclusion, these results indicated that Dab2 is positive regulator of the skeletal muscle development and differentiation both in vivo and in vitro.. |
