Dlk1 Is Necessary for Proper Skeletal Muscle Development and Regeneration Jolena N. Waddell1, Peijing Zhang 1 , Yefei Wen1, Sanjay K. Gupta2, Aleksey Yevtodiyenko3, Jennifer V. Schmidt4, Christopher A. Bidwell1, Ashok Kumar2, Shihuan Kuang1,5* 1 Department of Animal Sciences, Purdue University, West Lafayette, Indiana, United States of America, 2 Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky, United States of America, 3 Division of Oncology, School of Medicine, Stanford University, Stanford, California, United States of America, 4 Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America, 5 Center for Cancer Research, Purdue University, West Lafayette, Indiana, United States of America Abstract Delta-like 1homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression has been associated with muscle hypertrophy in animal models. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here we combine conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-kB and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy. Citation: Waddell JN, Zhang P, Wen Y, Gupta SK, Yevtodiyenko A, et al. (2010) Dlk1 Is Necessary for Proper Skeletal Muscle Development and Regeneration. PLoS ONE 5(11): e15055. doi:10.1371/journal.pone.0015055 Editor: Branden Nelson, Seattle Children’s Research Institute, United States of America Received August 17, 2010; Accepted October 15, 2010; Published November 29, 2010 Copyright: 2010 Waddell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was funded by National Institutes of Health grant #1R01AR060652-01A1 to SK and grant #HD042013 to JVS; Muscular Dystrophy Association grant #92478 to SK; United States Department of Agriculture - National Research Initiative Grant #2009-35206-05218 to SK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: skuang@purdue.edu Introduction The paternally expressed Delta-like 1 homolog (Dlk1) gene (Entrez Gene ID 13386) lies within the imprinted Dlk1 – Dio3 gene cluster on distal mouse chromosome 12 and encodes a transmembrane epidermal growth factor (EGF)-like protein[1]. The extracellular domain of Dlk1 can also be cleaved to generate a soluble form, called fetal antigen 1, which circulates as an abundant growth factor during development [2]. Another splice variant lacks the proteolytic cleavage site and remains constitutively membrane bound (Dlk1-C2). This membrane-bound form is most common in postnatal skeletal muscle [3,4]. Like many imprinted genes, Dlk1 is an important regulator of mammalian development and its expression level has dramatic effects on cellular proliferation and differentiation. Elevated Dlk1 expression is associated with many tumors including acute myeloid leukemia [5,6,7,8,9,10]. Accumu- lating evidence further indicates that Dlk1 is an important regulator of not only proliferation and differentiation of embryonic and adult stem cells but also functions to maintain the pluripotency of embryonic stem cells [4,11,12,13,14,15,16]. Recent reports indicate that Dlk1 may play important roles in skeletal muscle development. Transcript and protein levels of Dlk1 are highest in developing fetal muscle and taper off quickly after birth [2,17]. Interestingly, increased numbers of Dlk1+ mononu- clear cells and myofibers are reported in several myopathies including Becker and Duchenne muscular dystrophies which involves active muscle degeneration and regeneration [17]. Studies using transgenic mice over expressing Dlk1 [3,18] and callipyge mutation in sheep causing increased expression of Dlk1 [3,19,20,21] have also indicated that high levels of Dlk1 increase skeletal muscle mass in neonates. Callipyge sheep exhibit a significant increase in the size and proportion of type IIB fibers in muscles of the hind-quarters [22,23,24,25]. Muscle-specific over- expression of Dlk1 in mice yields similar results with an increase in total muscle mass and fiber diameter by six weeks of age [3]. Important roles of Dlk1 in muscle development is also evident by the observations that Dlk1-null mice show distinct phenotypes including increased mortality during late gestation and early neonatal development, skeletal malformations, and decreased growth rates accompanied by increased adiposity [26]. However, PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e15055