Role of Dlk1 in Myogenesis and Satellite Cells Figure 4. Dlk1 regulates satellite cell self-renewal and differentiation. A–D: Pax7 and MyoD expression in a representative cluster of myoblasts on a wild-type EDL fiber after 3 days of culture. Pax7+/MyoD2, Pax7+/MyoD+ and Pax72/MyoD+ cells represent self-renewing, proliferating and differentiating cells, respectively. E–H: A representative cluster of myoblasts on a cKO fiber cultured under identical culture conditions. I: The average number of cells per cluster. J: The percentage of cells expressing Pax7 and MyoD (note that most cells co-express Pax7 and MyoD; see K below). K: The proportion of cells at three different statuses: self-renewal (S–R; Pax7+/MyoD2), differentiation (Diff; Pax72/MyoD+) and proliferation (Prolif; Pax7+/MyoD + ). In I–K, n = 34 for WT, and n = 27 for cKO; * indicates p,0.05 compared to WT. doi:10.1371/journal.pone.0015055.g004 and the expression of sarcomere myosin heavy chain, revealed by MF20 immunofluorescence, is roughly 2.5 times higher in the Dlk1 over-expressing cells (Data not shown). These observations provide direct evidence that Dlk1 promotes myogenic differentiation. As extracellular ligands such as Dlk1 commonly exerts their effects on neighbor cells in a cell-contact dependent manner, we hypothesized that Dlk1 over-expression non-cell autonomously regulates myoblast proliferation and differentiation. To confirm that the observed effect of Dlk1 on myoblast differentiation is non- cell autonomous, we examined myoblast growth and differentia- tion in the presence of Dlk1 coated substrate. Wild-type myoblasts were allowed to proliferate and differentiate on a bed of Matrigel (BD Biosciences) containing vehicle control (Fig. 5N) or 500 ng/ml of recombinant Dlk1 protein (Fig. 5O). Cells differentiated in the presence of recombinant Dlk1 had reduced Ki67 expression (Data not shown) but formed significantly larger myotubes than cells grown under control conditions (Fig. 5P). These data, combined with the loss-of-function analysis, provide strong evidence that high levels of exogenous Dlk1 promote cell cycle withdrawal and differentiation of myoblasts into myotubes. Dlk1 upregulation correlates to myoblast differentiation and muscle regeneration in vivo Our data establish a non-cell autonomous role of Dlk1 in myogenic differentiation. To determine the physiological rele- vance of this observation, we examined whether Dlk1 upregulation correlates to myoblast differentiation. Indeed, Dlk1 protein expression, along with myosin heavy chain protein expression, is upregulated in newly differentiated myotubes 5 days after induction of differentiation (Fig. 6A). In addition, both mRNA (Fig. 6B) and protein (Fig. 6C) levels of Dlk1 were significantly upregulated 5 days after CTX-induced muscle regeneration in vivo. In contrast, Dlk1 protein levels were very low in both quiescent and activated satellite cells, but elevated in newly regenerated myofibers (Supplementary Fig. S1A&C). Similarly, cultured myoblasts expressed much lower levels of Dlk1 mRNA in relative to isolated myofibers or whole muscles (Supplementary Fig. S1D). Combined with our earlier results from Dlk1 cKO, these results indicate that Dlk1 upregulation is correlated and necessary for normal differentiation of muscle progenitor cells. Based on our findings and previous observations, we propose a model in which the timing of Dlk1 critically affect muscle regeneration after injury (Fig. 6D). Upon muscle injury, satellite cells are activated and proliferate. Dlk1 level in the whole muscle peaks at the height of satellite cell proliferation (Fig. 6A–C), allowing many of the proliferating myoblasts to differentiate and repair the muscle damage. As Dlk1 levels later drop[17], a portion of undifferentiated cells then return to the self-renewed state, or quiescence. The level of Dlk1 available on neighboring tissues during satellite cell activation determines, in a non-cell autono- PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e15055