Role of Dlk1 in Myogenesis and Satellite Cells transfected cells) exhibit little colocalized with Ki67 immunofluorescence. G: Percentage of cells displaying Ki67 staining in Control and Dlk1 OE cells (n = 3). H–J: Primary myoblasts transfected with an empty plasmid (H, control) or Dlk1 plasmid (I, OE) cultured for 3 days in growth medium and labeled with Ki67 (in red) and DAPI (in blue). The average Ki67 immunofluorescenc intensity was measured with Photoshop and normalized to DAPI intensity (J, n = 3 per treatment). K–M: Phase-contrast images of primary myoblasts differentiated for 48 hrs after transfected with either empty plasmids (K) or Dlk1 OE plasmids (L). M: Average pixel size of myotubes in Control and Dlk1 OE treatments as shown in H&I (n = 20 random tubes measured). N–O: Primary myoblasts grown on Matrigel plus vehicle control (N) and on Matrigel plus Dlk1 recombinant protein (O) after 3 days in differentiation medium. Green fluorescence (MF20) marks sarcomere myosin heavy chain and Blue is DAPI counterstaining for nuclei. P: The relative diameters of the resulting myotubes as shown in N & O were measured with Image J software. Control is the open bar (n = 66 myotubes) and Dlk1 recombinant protein treated cells are represented by the solid black bar (n = 66 myotubes). Asterisks in all bar graphs indicate p,0.05 compared to control groups by student t-test. doi:10.1371/journal.pone.0015055.g005 mous manner, the fate of the satellite cells in question. High levels of Dlk1 promote higher rates of differentiation while low levels trigger more self-renewing cells. However, our model does not exclude a potential cell-autonomous role of Dlk1 in myoblast differentiation. Finally, our Dlk1cKO mice had significantly higher levels of NF-kB activation, pro-inflammatory cytokines, and macrophage infiltration. Thus endogenous Dlk1 may also be required to limit the inflammatory response in regenerating muscles. Discussion Delta-like 1 (Dlk1) exists in both a cleaved circulating form and a constitutively membrane bound form and is crucial for the proper development of several tissues in mammals. However, the specific functions and signaling mechanisms of the predominantly membrane-bound form (Dlk1-C2) in skeletal muscle and muscle progenitor cells remain unclear. Previous reports have indicated Figure 6. Dlk1 up-regulation correlates to myogenic differen- tiation and skeletal muscle regeneration after injury. A: Representative Western-blot image showing that Dlk1 protein levels increase 5 days after serum-withdrawal induced differentiation of C2C12 myoblasts. B: The relative level of Dlk1 mRNA in the TA muscle revealed by quantitative Realtime PCR 5 days after cardiotoxin-induced regeneration (* indicates P,0.05; n = 18 for control TA; 6 for CTX treated TA). C: Dlk1 protein expression revealed by Western-blot in resting and regenerating TA muscles 5 days after cardiotoxin treatment. D: A model depicting how Dlk1 level varies during muscle quiescence and regeneration, which correlates to suppression of NK-kB activity and the onset of myoblast differentiation. doi:10.1371/journal.pone.0015055.g006 that muscle-specific over-expression of Dlk1 causes postnatal muscle hypertrophy in both mice and sheep [20,21,65,66]. However, the phenotype in sheep is complicated as the callipyge mutation also alters the expression of several other imprinted genes near the Dlk1 allele [19,20,21,66,67,68,69,70]. In addition, constitutive Dlk1 knockouts and over-expression are mostly embryonic or perinatal lethal[18,26,71]. This study was thus designed to elucidate the role of local non-circulating Dlk1 in skeletal muscle development and regeneration by creating a muscle-specific Dlk1 knock-out mouse (Dlk1 cKO). Floxed Dlk1 mice were crossed with a Cre driver allele under the control of the Myf5 promoter. Myf5 is an early critical muscle regulatory factor[32], so Dlk1-flox/Myf5-Cre mice have their Dlk1 gene excised during early embryonic development of muscle progenitor cells. By measuring the expression levels of Dlk1 in muscle-specific knock-out mice, it was observed that Dlk1 levels were only decreased by 35% in whole muscle compared to wild-type siblings. However, when Dlk1 was measured on isolated myofibers and myoblasts, the levels were greatly decreased in muscle-specific mutants, similar to data from Dlk1-null mice. Further investigation showed the Dlk1 protein is still detectable in mutant whole muscles originating from non-myogenic cells, mostly endothelial CD31+ cells. This is consistent with a recent study that reports many Dlk1+ cells found in adult muscle are mesenchymal or fibroblastic in nature, rather than myogenic [17]. However, our data do not indicate that Dlk1 is normally present in satellite cells, contrary to findings by Andersen and others [17]. Our findings suggest that Dlk1 from neighboring interstitial cells or myofibers [2,4] interacts with satellite cells to influence their physiological state. The total number of muscle fibers was significantly reduced in our conditional mutant mice. This may account for decreased total body mass that is observed when comparing Dlk1 cKO mice to wild-type littermates. This phenotype was not expected since the over-expression of Dlk1 does not change the number of myofibers present, only their size and metabolic functions [25]. One possibility is that by removing Dlk1 early in myogenic progenitor cells, the formation of primary and/or secondary myotubes in the embryo may be compromised. Because of fiber type changes observed in Dlk1 over-expression models, myosin heavy chain isoforms were measured by qPCR and immuohistochemistry in our Dlk1 cKO mutants. The mRNA levels of myosin heavy chain type IIB were significantly reduced in both the soleus and EDL muscles of mutant mice, while no other isoforms appeared to change. This is consistent with Dlk1over- expression models where the expression of type IIB myosin heavy chain was found to be increased in both sheep and mice [22,25,65]. This suggests that Dlk1 may play a role in either contraction speed or energy metabolism in the muscle. The role of Dlk1 in muscle regeneration was observed by challenging muscles with a cardiotoxin (CTX) injury. We found that muscle regeneration was impaired at several levels in the Dlk1 cKO mice. Myogenin levels and the number of nascent de novo PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e15055