fibers were significantly reduced, while cellular infiltrate and levels of pro-inflammatory cytokines were greatly increased. Increased levels of TNF-a have been shown to decrease nascent myofiber genesis [72,73] which along with IL-1b, activate the NF-kB signaling pathway [62,74,75]. We observed high levels of NF-kB in mutant regenerating muscle of Dlk1 cKO mice. Several studies have shown that NF-kB tightly controls myogenesis and specifically inhibits myofiber formation both in vitro and in vivo [61,76]. Our data show that lack of Dlk1 in skeletal muscle increases inflammatory response after injury and impairs regen- eration potentially through increased activation of NF-kB and expression of inflammatory cytokines. It is also possible that the enhanced inflammatory reaction to muscle injury is due to the poor regeneration of the Dlk1 cKO muscle. Furthermore, reduced phosphorylation of Akt in regenerating myofibers of Dlk12cKO mice suggests that Dlk1 may be enhancing myofiber regeneration though the activation of this kinase. Alternatively, inhibition in Akt phosphorylation could be a result of less myofiber regeneration in Dlk1 cKO mice. We provide direct evidence that Dlk1 function to regulate myogenic progenitor cells. Mice possessing the conditional Dlk1 mutation showed higher proportions of self-renewing cells at the expense of fewer proliferating cells (Fig. 4). In opposition, myoblast differentiation is accompanied by higher levels of Dlk1 protein (Fig. 6). More importantly, overexpression of Dlk1 inhibited myoblast proliferation and promoted their differentiation (Fig. 5). These results support a role of Dlk1 in the regulation of the proliferation and differentiation of satellite cells and provide a mechanism by which Dlk1 overexpression leads to muscle hypertrophy as seen in callipyge sheep and transgenic mice [3,18,19]. It remains unknown what signals regulates Dlk1 expression in regenerating and nascent myofibers, and the Dlk1 downstream effectors that affect myoblast proliferation and differentiation. Our results are consistent with the notion that Dlk1 acts as an inhibitor for Notch signaling [28,29,30]. Notch signaling is known to inhibit MyoD expression and myogenic differentiation, but enhance satellite cell proliferation and self-renewal[77,78]. Based on this notion, our Dlk1 conditional mutants should have elevated Notch signaling, which would suppress MyoD expression[79]. Our results confirmed this prediction. Conversely, over-expression of Dlk1 should suppress Notch signaling and promote myogenic differen- tiation, but inhibit myoblast proliferation[80,81]. Our overexpres- sion studies again support this notion. Interestingly, recent studies have also shown that Notch signaling interacts with the canonical NF-kB pathway. Notch-1 is able to promote the transcription of TNF-a [82,83], which in turn stimulates the phosphorylation of IkBa, leading to the activation of NF-kB signaling [83]. Notch-1 is also known to affect macrophage and cultured B cell signaling. Wild-type macrophages increase Notch-1 expression after an immune challenge, which then promotes the transcription of pro- inflammatory cytokines such as TNF-a [82]. In Notch-1-null mice, the immune response and NF-kB activity were three times lower in B-cells compared to wild-type cells [84].Thus, the increased immune response seen in our Dlk1 cKO injured mice is likely the result of increased Notch-1 signaling, which again support the notion that Dlk1 acts to suppress Notch signaling. In summary, our data provide novel insight into the mechanisms of action of Dlk1 in skeletal muscle. First we show that ablation of Dlk1 in the Myf5-derived myogenic cells leads to both developmental and postnatal growth/regeneration defects in myogenesis. As the conditional mutants should have normal levels of circulating Dlk1 which is mainly produced by the fetal liver, our results suggest that muscle specific membrane-bound Dlk1 is Role of Dlk1 in Myogenesis and Satellite Cells important for normal myogenesis. We next show that lack of Dlk1 in injured muscle increases the inflammatory response, increasing NF-kB signaling, and decreasing Akt activity. These shifts greatly decrease the ability of the injured muscle to regenerate appropriately. We further show that at progenitor cell level, Dlk1 expression inhibits myoblast proliferation but promotes myogenic differentiation. Therefore, the observed up-regulation of Dlk1 in wild-type muscles after injury may act to allow proliferating myoblasts to differentiate and fuse with existing fibers for muscle repair. When Dlk1 is not present in neighboring tissues, myoblasts are shifted towards the self-renewal state while increasing the inflammatory response, thus hindering muscle repair. These results add to our current understanding of the cellular and molecular mechanisms by which Dlk1 regulates skeletal muscle hypertrophy. Materials and Methods Mice and Animal Care Generation of Dlk1-floxed mice will be described elsewhere (Appelbe et al., in preparation). Briefly, loxP cassettes were inserted into the endogenous Dlk1 locus flanking exons four and five, and the mice were maintained in a C57BL/6 background. Myf5-Cre mice [85] were provided by Dr. Philip Soriano (Mount Sinai School of Medicine, New York, NY). Mice maintenance and experimental use were performed according to protocols approved by the Purdue Animal Care and Use Committee (PACUC protocol # 08-006). Muscle Injury and Regeneration Muscle regeneration studies were performed using cardiotoxin (CTX; Sigma-Aldrich, St. Louis, MO, USA) injections into the TA muscle. Mice were anesthetized using a ketamine-xylazine cocktail, then 25 mL of 10 mM CTX was injected into the right TA muscle. Muscles were then harvested either at 5 days post- injection for peak regeneration activity or 2–3 weeks post-injection to assess the completion of regeneration and repair. Whole Muscle Sections and Staining Whole muscles (TA, EDL, and soleus) were dissected and either frozen immediately in OCT compound or fixed. For fixation, muscles were placed in 4% PFA for 1 h, 100 mM glycine for h, then 30% sucrose overnight at 4uC. Fixed samples were then frozen in OCT compound. Frozen muscles were cut into 10 mm thick cross sections by a Leica CM1850 cryostat. Immunohisto- chemistry staining of muscle sections were performed as previously described[44]. Specific primary antibodies can be found in Supplemental Table S1. Fluorescent images were captured with a Coolsnap HQ CCD camera (Photometrics, USA) driven by IP Lab software (Scanalytics Inc, USA) using a Leica DM6000 microscope with a 20X objective (NA = 0.70). H–E staining images were captured by a Nikon D90 digital camera installed on a Nikon (Diaphot) inverted microscope. Monochrome images were processed and composited into color images using Photoshop (CS3) software. Single Fiber Culture and Staining Single myofibers were isolated from the EDL muscles by collagenase I (Roche Applied Science) digestion and tituration as previously described [86,87]. Suspended fibers were cultured on 60 mm horse serum-coated plates in DMEM media supplemented with 10% fetal bovine serum, 0.5% chick embryo extract, and 1% penicillin-streptomycin for three days. Fibers were then fixed in PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e15055