several key questions remain unresolved: 1) the relative role of membrane bound versus cleaved forms in muscle development and growth is unclear; 2) as Dlk1 can be cleaved and circulate, it is unknown whether muscle specific Dlk1 expression is necessary for normal muscle development and growth; 3) what role does Dlk1 play in postnatal muscle regeneration and muscle progenitor cells? In addition, the mechanisms by which Dlk1 regulates skeletal muscle development and hypotrophy are poorly understood. Due to the EGF-like domain in Dlk1, it is classified into the same family as the Notch receptors and ligands including Delta and Serrate (Jagged) [10,27]. Unlike Delta and Serrate, however, Dlk1 lacks the DSL (delta/serrate/lag) domain known to be essential for activation of Notch receptors. Therefore, Dlk1 is thought to interact with the Notch receptors through its EGF repeats as an antagonist and down-regulate signaling through Notch in Drosophila and Notch-1 in mammalian cell cultures [28,29,30]. Activation and suppression of Notch receptors by Delta occur in both cis (Delta suppresses Notch on the same cell; cell autonomous) or trans (Delta from one cell activates Notch on a neighboring cell; non-cell autonomous) manner [31]. Such distinction in signaling among progenitor cells would fit in the model of asymmetric cellular commitment seen in activated satellite cells in muscle [32]. Activated Notch signaling inhibits the formation of muscle progenitor cells in Drosophila [33,34,35] and delays the expression and activation of MyoD and myogenin, markers of muscle differentiation, in mammals [36,37]. This suggests a model in which Dlk1 facilitates muscle differentiation through dampening of Notch signaling. However, a recent study suggests that Dlk1 does not interact with Notch1 receptor nor requires Notch activation to exert its effect on preadipocyte differentiation[38]. Satellite cells are muscle-specific stem cells that lie quiescent beneath the basal lamina of adult myofibers until needed for muscle repair. Following an injury, satellite cells proliferate and incorporate their nuclei into existing fibers while still maintaining their stem cell population. Donor satellite cells have even been shown to engraft onto recipient fibers to repair injured myofibers and ameliorate disease progression [32,39,40,41,42]. The status of a satellite cell, whether self-renewing, proliferating, or differenti- ating, can be determined by the expression of Pax7 and MyoD [43,44,45,46,47,48,49,50,51,52]. We use these markers to deter- mine the molecular mechanisms by which Dlk1 regulates fate of satellite cells during muscle development/regeneration. To investigate the requirement of muscle specific Dlk1 in myogenic development and postnatal muscle repair, we generated a conditional Dlk1 knock-out mouse using a Dlk1-floxed allele together with a Myf5-Cre driver. Since Myf5 is the earliest expressed myogenic regulatory factor in developing embryos and is required for myogenic determination [53,54], this model is useful to determine how Dlk1 function in both myogenic progenitor cells and mature skeletal muscles. Our results show that muscle-specific depletion of Dlk1 leads to defective muscle development, elevated inflammatory responses, and delayed muscle regeneration. We further provide evidence that Dlk1 non-cell autonomously affects satellite cell fate choice between self- renewal and differentiation. Results Defective muscle formation, growth retardation, and altered myosin heavy chain gene expression in muscle- specific Dlk1 mutant mice The role of membrane-bound Dlk1 in skeletal muscle was investigated by creating a conditional mutant mouse model. We used the Myf5-Cre mouse to inactivate Dlk1 in the myogenic Role of Dlk1 in Myogenesis and Satellite Cells lineage [53,54] of Dlk1flox mice. As Dlk1 is an imprinted gene expressed only from paternal allele [1], paternal heterozygous Myf5-Cre/Dlk1+(m)/flox(p) displayed identical Dlk1 expression profiles and phenotypes as the homozygous Myf5-Cre/Dlk1flox/ flox alleles (data not shown). Thus, Dlk1flox/+ males were crossed with Myf5-Cre females to produce paternal heterozygotes for muscle-specific Dlk1 knockout (henceforth Dlk1 cKO). By performing qPCR assays we found ,35% reduction in Dlk1 expression in whole muscles of Dlk1 cKO, suggesting that Dlk1 expressed by myofibers only accounts for about one-third of the total Dlk1 mRNA expressed by the whole muscle (Fig. 1A). These results are in agreement with our immunohistological results that Dlk1 is mainly expressed by interstitial non-myogenic cells within the muscle (Supplementary Fig. S1). To confirm muscle-specific knockout of Dlk1, we isolated single myofibers from EDL muscles and quantified Dlk1 expression, which was reduced by ,90% in the Dlk1 cKO myofibers (Fig. 1A). The residual (10%) Dlk1 expression detected in isolated Dlk1 cKO myofibers probably originated from contaminating interstitial cells attached on the fibers[44]. Furthermore, Dlk1 levels in Dlk1 cKO brown adipose tissue (BAT) were reduced by 80%, again in agreement with our recent report that the brown adipose tissue is derived from a Myf5- lineage[55]. In contrast, white adipose tissue, known to be derived from Myf5-independent lineages[55], exhibited no changes in Dlk1 expression. These results confirm specific deletion of Dlk1 in muscle and brown fat lineage mediated by the Myf5-Cre allele. The Dlk1 cKO mice were born at expected Mendelian ratios (Expected ratio: 25%; observed ratio 25.963.9%, n = 17 litters) and appeared to be healthy and behaviorally normal. However, notable phenotypic differences were observed in the Dlk1 cKO mice compared to wild-type siblings. First, the mutants displayed a significant reduction in body weight (Fig. 1B); this reduction appeared to be more prominent at older age (11% reduction in old vs 5% in young mice, however p = 0.34). The brown fat mass also decreased by 20% in the Dlk1 cKO mice (Fig. 1C). To further determine if the observed body weight loss was mainly due to reduced muscle mass, we compared the number of muscle fibers in representative fast (EDL, Fig. 1D) and slow (soleus, Fig. 1H) muscles. Indeed, there is a roughly 25% reduction in total myofiber numbers in the cKO in both muscle types (Fig. 1E & I), which likely contributes to the 5–10% decrease in body mass, as skeletal muscles generally account for about 40% of total body weight. As myofiber numbers are known to be fixed at birth[56], the reduced number of myofibers in cKO reflects defects in embryonic myogenic development. Together, these observations suggest that our Dlk1conditional mutation leads to defective muscle development and postnatal growth retardation. Over-expression of DLK1 in Callipyge sheep muscle is linked to a switch in fiber type towards fast twitch glycolytic fibers that express the myosin heavy chain (MyHC) IIB gene. We therefore examined if the deletion of Dlk1 leads to fiber type switching. A marked reduction in mRNA levels of MyHC IIB was detected in the Dlk1 cKO mice in both EDL and SOL muscles (Fig. 1F & J), whereas the expression of other MyHC genes were not affected (not shown). MyHC isoform-specific monoclonal antibody labeling of EDL and SOL muscles, however, did not indicate any differences in the proportion of each fiber type (Fig. 1G & K). Therefore, muscle-specific ablation of Dlk1 appears to reduce the levels of fast type IIB MyHC, but does not lead to fiber type switching in mice. Delayed muscle regeneration in Dlk1 cKO To investigate how Dlk1 deficiency may affect adult myogen- esis, The tibialis anterior (TA) muscles of Dlk1 cKO and wild-type mice were given intramuscular injections of CTX and the PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e15055