Role of Dlk1 in Myogenesis and Satellite Cells Figure 2. Muscle-specific Dlk1 cKO results in impaired muscle regeneration after injury. A–F: Morphology of TA muscle 1 week after cardiotoxin injury in WT (A–C) and cKO mice (D–F). A, D: Fluorescent immunostaining of mouse IgG showing injured fibers in red and Dapi nuclei staining in green. B, E: Brightfield images showing increased fibrosis and scarification (Dark signal due to poor light penetration) in cKO mutant muscles. C, F: H–E staining showing poor organization and increased infiltration of non-myogenic cells in mutant muscles. G: Myogenin mRNA levels increase after injury, but cKO mice have an impaired myogenin response (n = 4). H: Total number of nascent fibers (n = 7 pairs) are decreased in cKO injured muscles. I: Levels of phosphorylated Akt are decreased in cKO muscle compared to WT control and injured muscles. doi:10.1371/journal.pone.0015055.g002 regeneration in response to CTX-mediated injury [63]. We therefore sought to investigate whether depletion of Dlk1 affects the activation of NF-kB transcription factor in regenerating muscles. DNA-binding activity of NF-kB in regenerating muscle was found to be markedly higher in Dlk1 cKO mice compared to wild-type mice (Fig. 3A). The increased activation of NF-kB pathway was also evident by our results that the levels of phosphorylated IkBa was up-regulated in regenerating muscle of Dlk1cKO mice compared to those of wild-type mice (Fig. 3B). Furthermore, the expression of NF-kB-regulated pro-inflammato- ry cytokines IL-1b and TNF-a was also significantly higher in myofibers of Dlk1cKO mice compared to wild-type mice in response to injury (Fig. 3D–E). Both TNF-a and IL-1b have been previously shown to inhibit myogenesis [62,63,64]. Collectively, these data suggest that the loss of Dlk1 exacerbates the inflammatory response and augments the expression of inflam- matory cytokines which may be responsible for the reduced myofiber regeneration after injury. Dlk1 ablation leads to reduced MyoD expression and facilitates self-renewal of activated satellite cells The defective muscle regeneration suggests that our muscle- specific Dlk1 mutation may also affect the normal function of satellite cells, which underlies muscle regeneration. To investigate this possibility, the potential effects of Dlk1 on satellite cell self- renewal and differentiation were assessed ex vivo by activating quiescent satellite cells attached to single EDL myofibers in culture. In this model, satellite cells initially only express Pax7, then activate MyoD, and enter the cell cycle. Proliferating cells then either down- regulate Pax7 in order to differentiate, or down-regulate MyoD and self-renew[43,45,51]. After 3 days in culture, distinctive clusters of myoblasts are readily detectable on myofibers. Myoblast clusters were stained for Pax7 and MyoD so that Pax7+/MyoD 2 , Pax7+/ MyoD+, and Pax72/MyoD+ cells represent self-renewing, prolifer- ating, and differentiating cells, respectively (Fig. 4A–H). The average number of cells per cluster was not significantly different between wild-type and conditional mutants (Fig. 4I). In PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e15055