Role of Dlk1 in Myogenesis and Satellite Cells Figure 1. Myf5-Cre mediated mutation of paternal Dlk1(cKO) results in defects in muscle formation and growth. Asterisks in all graphs denote p,0.05compared to wild-type (WT) controls. A: Quantitative PCR confirming muscle-specific Dlk1 knockout in cKO mice in whole muscle, myofibers, myoblasts and BAT. B: Relative body weight (BW) of WT (n = 17, 9, 4 for all age, 2–4 month and 10–12 month mice, respectively) and cKO (n = 18, 9, 5) littermates. C: Relative mass of BAT of WT (n = 3) and cKO (n = 3) mice. D–K: Muscle fiber composition in representative fast-twitch (EDL) and slow-twitch soleus (SOL) muscles revealed by MyHC isoform-specific antibodies and qPCR. D: MyHC isoform staining of representative EDL. Total myofiber number for EDL(E) and SOL (I) muscle in WT and cKO (n = 4 pairs). Myosin heavy chain (MyHC) IIB gene expression in EDL (F) and SOL (J;n = 3 pairs) muscles. Percent of each MyHC isoform by immunostaining in EDL (G) and SOL (K; n = 3 pairs). doi:10.1371/journal.pone.0015055.g001 regenerated muscles were examined at different time points. Regeneration of the TA muscle was considerably impaired in the Dlk1 cKO at 5–7 days after injury, a time point at which regeneration peaked in the wild-type mice. Skeletal muscle regeneration involves the repair of existing degenerated fibers and de novo formation of new fibers. Existing fibers undergoing active repair can be readily identified by their large diameter with centrally localized nuclei and non-specific IgG binding due to immune cell infiltration (Fig 2A). De novo formed new fibers are considerably smaller in diameter with central nuclei. While wild- type muscles regenerated uniformly with little fibrosis and scarification (Fig. 2A–C), the Dlk1 cKO muscles were poorly regenerated with extensive fibrosis, scarification, and interstitial space occupied by massive cellular infiltration (Fig. 2D–F). The defective regeneration of Dlk1 cKO fibers is substantiated by a significant decrease in myogenin mRNA expression levels (Fig. 2G) and a roughly 25% decrease in nascent de novo fiber formation (Fig. 2H) five days after injury. In addition, phosphor- ylated Akt (the activated form) levels were decreased in the cKO (Fig. 2I), suggesting an impairment of the Akt/mTOR signaling pathway that is known to promote protein synthesis and myotube hypertrophy [57]. This observation is consistent with our previous finding that Akt signaling is enhanced in DLK1 over-expressing callipyge sheep [58]. Together, these data suggest that Dlk1 is necessary for proper skeletal muscle regeneration in adult mice. Elevated inflammatory response to injury in Dlk1 cKO muscles As muscle injury causes an inflammatory immune response within the damaged tissue [59],we examined whether aberrant inflammatory responses contributed to the defective regeneration of the Dlk1 cKO muscles. The increased interstitial nuclei density in regenerating cKO muscles (Fig. 2F) indicates excessive infiltrated macrophages, which was confirmed by the high mRNA levels of CD68 (Fig. 3C), a cell surface marker for macrophages. Nuclear factor kappa B (NF-kB) is a major pro-inflammatory transcription factor controlling the expression of a plethora of genes involved in inflammation and immune responses [60]. Several published reports have suggested that NF-kB inhibits myogenesis at multiple levels including suppression of MyoD [61,62]. Moreover, it has been found that muscle-specific inhibition of NF-kB dramatically improves skeletal muscle PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e15055