Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

Sindhu Subramaniam; Prethish Sreenivas; Sirisha Cheedipudi; Vatrapu Rami Reddy; Lingadahalli Subrahmanya Shashidhara; Ravi Kumar Chilukoti; Madhavi Mylavarapu; Jyotsna Dhawan

doi:10.1371/journal.pone.0065097

Abstract

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G₀ myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G₀ C2C12 myoblasts to isogenic G₀ fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G₀ is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.

Citation: Subramaniam S, Sreenivas P, Cheedipudi S, Reddy VR, Shashidhara LS, Chilukoti RK, et al. (2013) Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest. PLoS ONE 8(6): e65097. https://doi.org/10.1371/journal.pone.0065097

Editor: Huating Wang, The Chinese University of Hong Kong, China

Received: October 21, 2012; Accepted: April 23, 2013; Published: June 3, 2013

Copyright: © 2013 Subramaniam 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: This work was supported by CSIR doctoral research fellowships to SS, PS and SC, and grants from Govt. of India Dept. of Biotechnology, CSIR Network Program on Cell and Tissue Engineering, Indian Council of Medical Research, a Wellcome Trust International Senior Research Fellowship to JD, and core support from the Dept of Biotechnology to the Institute for Stem Cell Biology and Regenerative Medicine. 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.

Introduction

Most cells in adult mammals do not divide. During tissue formation, cells exit the cell cycle either permanently or temporarily: for example, skeletal muscle although largely composed of post-mitotic myofibers, harbours rare dormant satellite stem cells. During regeneration of damaged muscle, satellite cells (SC) break quiescence and return to active proliferation. Subsequently, the type of cell cycle exit undertaken by the SC progeny has different consequences. One pathway is co-ordinated with the activation of tissue-specific genes and fusion to form multinucleate contractile myofibers, which restore tissue form and function but cannot return to active cell division. The other pathway, associated with suppression of differentiation, is a transient exit that permits replenishment of the SC reserve and is therefore central to SC function [1]. The two distinct modes of cell cycle exit have implications for maintaining the balance of cell types (differentiated vs. stem cell) in adult tissues, and deregulation of cellular quiescence programs may underlie pathological states such as cancer and degenerative disease.

Adult stem cells cycle rarely (classically revealed in label retention assays), and may spend much of their lifespan in G₀, yet quiescence is the least understood aspect of the cell cycle. Reversibility of G₀ requires programs beyond those that control the cell cycle per se [2] [3]. Growing evidence suggests that exit into G₀ is not a default state resulting from an absence of growth promoting signals, but is actively regulated [4]. The mechanisms that regulate the quiescence program are likely to operate not only at the level of signaling, but also transcriptional and chromatin modulation to maintain cellular identity.

The core transcriptional program of quiescence has been defined in hematopoietic SC [5] [6] and fibroblasts [2]. As with the cell division cycle [7], analysis of G₀ in yeast [8] [9] provides a conserved framework for understanding quiescence in mammalian cells. Distinct pathways may control the entry into and exit from quiescence [8] [10] and control of this dormant state is emerging as a complex program, with implications beyond arrest [2]. Thus, quiescence-induced programs may include a novel class of tumor suppressor genes that not only enforce cell cycle exit but also control other attributes of ‘hibernating’ cells. Beyond promoting survival under conditions of reduced/altered metabolic activity, quiescence factors would ideally also maintain a state of signal-responsiveness for cell cycle re-entry. Quiescent adult muscle stem cells would need to suppress overt differentiation, yet maintain lineage memory so as to follow the appropriate tissue-specific pathway when activated [11].

Molecular correlates of quiescence are difficult to study comprehensively in vivo: while expression profiling of freshly isolated muscle SC has been reported [12] [13], the G₀ state itself has not been accessed, as isolation from their niche breaks quiescence, forcing cells to enter G₁. The current understanding of quiescence biology has benefited from culture systems that generate homogeneous populations of reversibly arrested muscle cells or “G₀ myoblasts” [1].

Mitogen deprivation of asynchronous C2C12 cultures triggers irreversible cell cycle arrest, fusion and differentiation into multinucleated myotubes, [14] [15]. In serum-deprived cultures, a small proportion (∼20%) of cells resist differentiation and enter reversible quiescence while suppressing MyoD expression [16]. These ‘reserve cells’ have been used to model satellite cell growth control, but little is known about their formation under conditions that cause differentiation of the bulk of myoblasts. By contrast, during suspension culture, >99% of myoblasts exit the cell cycle in an undifferentiated state despite the presence of saturating concentrations of growth factors [17] [18]. Importantly, this anchorage-dependent arrest is synchronously reversed upon restoration of surface contacts in >98% of cells. Our previous studies have demonstrated that reversibly arrested C2C12 myoblasts model several aspects of satellite cell behavior [17] [18]. Firstly, myoblasts arrest in G₀ as evidenced by the absence of DNA synthesis, a 2C DNA content and suppression of growth-associated genes. Secondly, expression of MyoD and Myf5 is suppressed and differentiation-dependent genes such as Myogenin, myosin, and muscle creatine kinase are not induced. Thirdly, arrested myoblasts are synchronously activated out of G₀, sequentially express MyoD and Myf5 in G₁ and enter S phase. Finally, in addition to the myogenic regulators, several genes implicated in SC arrest [19], commitment [20], and activation [21] [1] are similarly regulated during reversible arrest in culture. Taken together, these findings indicate that reversible arrest in suspension culture involves the regulation of some key genes implicated in satellite cell function in vivo [18] [1] Further, we have used this homogenously arrested, synchronized system to identify genes that are induced in G₀ [18] [22].

Two important concepts emerged from these studies. Firstly, genes such as the RNA-binding protein TTP and the chemokine LIX which were strongly up-regulated in quiescent C2C12 myoblasts are expressed in Pax7+ satellite cells in normal skeletal muscle in vivo [18]. Secondly, genes identified in C2C12 myoblasts on the basis of quiescence-induced expression also function in key regulatory pathways: for example, knockdown of p8/Nupr1 a small chromatin architectural factor induced in G₀ myoblasts leads to hastening of the cell cycle, supporting an anti-proliferative role [22]; while knockdown of MLL5, a histone methyltransferase induced in G₀ myoblasts leads to loss of myogenic memory and failure of differentiation upon return to the cell cycle [11]. Thus, substantial evidence supports the hypothesis that genes induced in suspension-arrested G₀ C2C12 myoblasts are functionally involved in the quiescence program, and warrants further exploration at a global level.

Transcriptomal profiling of quiescent fibroblasts, lymphocytes and HSC [2], [4], [5] have substantially increased our understanding of the global networks that control this important “out-of-cycle” state. Despite the identification of common core programs in G₀, expectedly featuring tumor suppressors and inhibitors of differentiation, direct comparisons of different cell types in quiescence have not been reported, leaving a gap in our understanding of key mechanisms. Further, no direct comparisons have been made of a single cell type induced to different states of arrest. To address these open questions, we have used closely related cell types (myoblast and fibroblast), as well as compared a single cell type in different states of arrest (reversible vs. irreversible).

In this study, we describe gene expression profiling of reversibly arrested C2C12 myoblasts and C3H 10T1/2 fibroblasts in culture. We define a signature of genes associated specifically with G₀ and by comparison of G₀ in two different cell types, reveal the existence of a tissue-specific quiescence program. We also distinguish reversible quiescence in myoblasts from post-mitotic arrest in myotubes. Our analysis shows that Wnt pathway induction is associated with the quiescent state in myogenic cells, implicates Wnt signaling in the choice between reversible and irreversible arrest/differentiation and finally, identifies two new quiescence-induced regulators of Wnt signaling.

Materials and Methods

Cell Culture

C2C12 myoblasts [23] [15], were obtained from H. Blau (Stanford) and sub-clone A2 [18] used in all experiments. Myoblasts were maintained in growth medium (GM: DMEM with 20% FBS). C3H10T1/2 cells (a multipotent mesenchymal line) obtained from H. Blau were maintained in DMEM+10% FBS.

Differentiation was induced in cultures at ∼80% confluence after washing with PBS and incubation in differentiation medium (DM: DMEM with 2% horse serum), replaced daily for 3–5 days.

G₀ synchronization of myoblasts was achieved using suspension culture as described [18]. Briefly, sub-confluent cultures were harvested and cultured as a single cell suspension (10⁵ cells/ml) in semi-solid DMEM containing 1.3% methyl cellulose, 20% FBS (>98% of cells arrest in G₀ by 48 hrs). Fibroblasts were suspension-arrested in DMEM+10% FBS containing 1.3% methyl cellulose as described for myoblasts.

Reserve cells were generated by the method described previously [24]. Briefly, dense cultures of myoblasts were incubated in DM for five days. Myotubes were quantitatively removed by mild trypsinization, and remaining (adherent) quiescent mononuclear reserve cells isolated by complete trypsinization and replated for 0–24 hrs.

Transfection

C2C12 myoblasts plated on cover slips (for imaging) or 60 mm dishes (for FACS) were transfected as previously described [11]. Where indicated, cells were treated with rmWnt3A (50 ng/ml, R&D Systems).

Luciferase assays were performed as described [25] on cells plated in 24 well plates and transfected with Topflash or pGL3luc control plasmids, along with pSV2-lacZ to normalize for transfection efficiency using β-gal assays.

Immunofluorescence

Cells plated on cover slips were fixed, and processed as described [11] for confocal microscopy (antibody details in Materials and Methods S1). Images were adjusted minimally for brightness and contrast using global settings applied to the whole image and assembled using Adobe Photoshop.

Microarray Analysis

Hybridization.

RNA was isolated as described [18] from C2C12 myoblasts proliferating asynchronously (MB), G₀ synchronized (G₀ MB), 3 day differentiated myotubes (MT) or G₀ FB (G₀FB; C3H10T1/2), Cy3- or Cy5-labelled cDNA synthesised using Superscript II, and used in competitive hybridization experiments as described [11]. Each chip [NIA15K mouse spotted cDNA arrays (University Health Network, Ontario)] contains ∼15,000 mouse cDNAs and ESTs [26], spotted in duplicate along with controls. At least 2 arrays (each with duplicate spots) were used for each sample pair, including a biological replicate and a dye reversal experiment. Thus for each gene ID, data was collected from 4–6 replicate hybridized elements.

Array informatics and statistics.

A suite of statistical and image analysis programs was used for analysis and the MIAME-compliant data is available at GEO (www.ncbi.nlm.nih.gov/geo/- Accession # GSE33676, GPL14883, GPL14884). Array Vision software was used for feature extraction, background subtraction, detection limit and intensity calculations. Normalization to remove systematic bias was done using locally weighted linear regression (LOWESS), flip-dye analysis was performed using MIDAS software (from TIGR), statistical significance analysis (false discovery rate <4%) was performed using SAM (Stanford Microarray Resource). A cut-off of 1.6-fold (Normalized Log Ratio [NLR] 0.67) was used to designate genes as up- or down-regulated. Hierarchical clustering was done using the TIGR TmeV program; GO annotations and other details for all genes were obtained from the Stanford SOURCE database.

Northern blot analysis was performed as described [18] using 10 µg of total RNA and ³²P-labeled probes generated by PCR of mouse cDNA clones derived from the NIA 15K clone set using SP6 and T7 primers.

Cell cycle analysis.

Flow cytometric analysis of DNA content was done using propidium iodide staining as described [11] and analysed on a FACS Vantage using CelQuest software.

Quantitative Real-Time RT-PCR for transcript analysis was performed on an ABI 7700HT PCR machine as described [11]. A list of primers is provided in Materials and Methods S1.

Wnt superarray.

1 µg of total RNA isolated from MB, G₀ MB or MT was analysed by Q-RT-PCR in a pre-spotted 384 well format according to manufacturer’s instructions (Superarray, Qiagen). The superarray contains primers specific to 84 different genes in the Wnt pathway.

Knockdown analysis.

Myoblasts were transfected with si or shRNAs (control si/sh RNA, Dkk3sh and Rgs2sh) as described [11]. Transfected cells were purified by FACS-sorting (see below) and extent of knockdown confirmed by Q-RT-PCR and western blot analysis. Details of knockdown oligos and primers are available as Materials and Methods S1.

Chromatin Immuno-Precipitation (ChIP) assay.

ChIP analysis was performed essentially as described earlier [11]. Briefly, C2C12 cells were grown to 80% confluency in GM, washed with PBS and collected in cell dissociation buffer (Sigma), re-suspended in GM and chromatin complexes cross-linked with HCHO (added to a final concentration of 1%). Cross-linking was quenched by addition of PBS, cells were washed by centrifugation through ice-cold PBS, the pellet re-suspended in SDS lysis buffer and processed for sonication. Sonication conditions (available on request) were standardized for different cellular states since nuclear condensation/chromatin configuration differs and sonicated samples stored at −80 as aliquots. Pre-clearing, antibody pull-down, elution, reversal of cross-linking and DNA isolation was performed as per manufacturers instructions (ChIP kit, Millipore). For each IP 2–5 µg of primary antibody was used. Control pull-downs used IgG (5 µg) to assess specificity.

Quantitative Real-time PCR analysis on ChIP samples was performed on an ABI 7900HT cycler (Applied Biosystems) as described [11]. The ct value of input was subtracted from IP and Control IgG sample and the resulting IP value was normalized again with the resulting control IgG value for calculation of fold enrichment in each sample by 2^(−ΔΔct) method [ie 2^{−[(IP-input)−(IgG−input)]}]. The values obtained for each gene represent normalized fold enrichment of the IP protein over input and Control IgG. These values are represented as fold change with respect to the level in myoblast (Mb) samples, so that different genes and different samples can be compared directly. ChIP primers used for Myogenin cover a genomic region including the well-characterized Mef2 binding site and the −130 bp upstream E-box [27]. The Myf5 ChIP primers were designed to cover the −57 kb upstream epaxial enhancer which is a characterized Myf5 cis regulatory region known to be important for Myf5 expression in limb bud [28]. Details of primers are provided in Materials and Methods S1.

Colony assays (CFU).

Control and treated MB were held in suspension for 48 hrs (with or without Wnt3a (R&D systems cat# 1324-WN) or sFRP2 (gift from Dr. Arun Dharmarajam, School of Anatomy & Human Biology The University of Western Australia), recovered from methocel, counted, re-suspended in GM without factors, plated at clonal density (400 cells/150 mm dish) and cultured for 7 days. Colonies were stained with methylene blue for counting. shRNA transfected cells were identified using a co-transfected EGFPC1 (Invitrogen, USA) plasmid, GFP+ cells were FACS-sorted and analysed by plating in CFU assays.

Results and Discussion

Model System

The term G₀ is widely used to refer to the non-replicative state in which either post-mitotic cells or reversibly arrested cells exist. Here, we use G₀ to refer exclusively to reversible arrest. Mitogen deprivation of adherent myoblasts (MB) triggers arrest, fusion, sustained expression of the myogenic regulatory factor (MRF) MyoD, activation of Myogenin (MyoG) and differentiation into multinucleated myotubes (MT) (Fig. 1A) [29]. By contrast, non-adherent culture of MB in mitogen-rich methyl-cellulose induces cell cycle exit in an undifferentiated state, and loss of MyoD expression [17] [18]. These MyoD-negative MB fail to incorporate BrdU (Fig. 1A), possess a 2C DNA content (Fig. 1B), and express Pax7 and specific cell cycle inhibitors (Fig. 1C,D). Differentiated MT enter a post-mitotic state that cannot be reversed by mitogens [14], whereas suspension-arrest is reversed upon replating on adhesive surfaces [17] (Fig. 1E). Several criteria confirm that suspension-arrested MB enter G₀: absence of DNA synthesis, a G₁ DNA content (an un-replicated genome), gross transcriptional and translational suppression, absence of both myogenic and growth-associated gene expression, and the extended kinetics of S-phase re-entry in comparison to exponentially cycling populations [18] [17]. Cell cycle re-entry restores differentiation competence, as MyoD is reactivated in G₁ (Fig. 1F), [25] [30], but overt differentiation needs additional cues [31] [32].

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Figure 1. C2C12 myoblasts enter alternate states of arrest.

(A) Asynchronous proliferating myoblasts (MB) were held in suspension in mitogen-rich media to induce quiescence (arrest in an undifferentiated mono-nucleated state; G₀ MB), or shifted to mitogen-poor medium for 96 hrs to induce differentiation (arrest coupled with fusion into multinucleated myotubes; MT). Actin staining (Oregon Green-Phalloidin, top panel) reveals the distinct morphology of these 3 states. Nuclei are detected with Hoechst 33352. DNA synthesis was analyzed by detection of BrdU incorporated in a 15-minute pulse (middle panel)- ∼40% of cycling MB are labeled, while less than 1% of G₀ MB synthesize DNA. In differentiated cultures (MT), only residual mono-nucleated cycling cells (5–10%) incorporate label whereas myotube nuclei do not. Expression of determination factor MyoD can be detected in ∼50% of proliferating MB, is lost in G₀ but sustained in MT (lower panel). (B) FACS analysis of DNA content of cycling (Asynchronous MB) and 48-hr suspension-cultured populations confirms that >97% of cells in suspension (G₀ MB) possess a 2C DNA content while >40% of cells in an asynchronous culture have replicated their genomes (representative graphs from 4 independent experiments). (C) Muscle transcription factor expression distinguishes alternate states of arrest. Q-RT-PCR analysis of specification/survival factor Pax7, determination factor MyoD, and early differentiation marker Myogenin (MyoG) in asynchronous proliferating myoblasts (As), suspension-arrested MB (G₀) and differentiated MT (MT). Values represent normalized fold differences between GAPDH (control) mRNA and myogenic mRNAs in each sample (n = 3). (D) Cell cycle inhibitor expression distinguishes alternate states of arrest-G₀ MB and MT express different combinations of cyclin-dependent kinase inhibitors p21/p27 and Rb-related p130. Immuno-detection of p21, p130 or p27 in MT and G₀; p21 (green) co-localizes with MyoG (red) in all nuclei of differentiated MT but neither factor is expressed in G₀; p130 is specific to G₀ and p27 is expressed in both G₀ and MT. Data depicted is representative of three independent experiments. (E) G₀ arrest is reversible. DNA synthesis in asynchronous MB (Mb), 48-hour suspension cultures (G₀) and G₀ MB replated for 6 or 24 hours (R6, R24). BrdU detected as in Fig. 1A. Values represent mean+SEM (n = 3). (F) MyoD expression is suppressed in G₀ and restored during early reactivation. Asynchronous cultures (A) were arrested in G₀ (48 hrs) and reactivated by replating for 2 to 24 hrs (R2–R24). MyoD expression detected as in Fig. 1A. Values represent mean+SEM (n = 3).

https://doi.org/10.1371/journal.pone.0065097.g001

Microarray Profiling of Arrested Myoblasts and Fibroblasts: Experimental Design and Data Analysis

Our earlier studies [11] [18] distinguished the state of G₀ from that of differentiated myotubes with respect to individual genes, laying the foundation for a genome-wide analysis.

To directly assess alternate programs of cell cycle exit, we compared reversibly arrested and post-mitotic (differentiated) C2C12 cells by microarray profiling using cDNA arrays (Fig. 2A): To identify transcripts induced in quiescence, we compared G₀ MB with asynchronously growing MB. To enrich genes induced by cell cycle exit in an undifferentiated state, we compared G₀ MB with permanently arrested differentiated MT. To enrich genes associated with differentiation, we compared MT with MB. Finally, to explore tissue-specificity in G₀, we compared G₀ MB with isogenic G₀ ‘fibroblasts’ (FB; multipotent C3H10T1/2 cells, which like C2C12, are derived from C3H mice). Microarray data can be accessed at GEO with accession number GSE33676. Genes differentially regulated between the 4 sample pairs are shown in Fig. 2A. Among the 15,000 gene elements surveyed, 1157 were induced >1.6-fold in G₀ (q value [false discovery rate] 4%), supporting the notion that quiescence involves large-scale reprogramming of the transcriptome.

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Figure 2. Microarray analysis reveals distinct genetic programs in reversible and irreversible arrest.

(A) Schematic of microarray loop experiment. RNA isolated from G₀ MB was analyzed by 2-color competitive hybridization to NIA15K mouse cDNA arrays. Pair-wise analysis: G₀ MB vs. either proliferating MB, differentiated MT or G₀ FB. In each pair, genes consistently up- or down-regulated (>1.6 fold, normalized log ratio (NLR) 0.67, false discovery rate 4%) were selected for further analysis. The numbers of genes regulated between each pair of samples are shown: induced (red), suppressed (green). Expression of key regulators characteristic of the different samples is indicated, confirming the identity of each cellular state. (B) Hierarchical clustering (MEV software, TIGR) shows the relatedness of the myoblast and fibroblast sample pairs drawn from independent biological experiments including a dye reversal test. Based on the total transcriptional response, genes up-regulated in G₀ MB fall into 3 broad categories-genes specifically induced in G₀ MB (the largest number), genes induced in both G₀ MB and MT but down-regulated in G₀ FB, and genes induced in G₀ MB and G₀ FB but not in MT. (C) In muscle cells, common features of two types of cell cycle exit (reversible vs irreversible) are overshadowed by differences. Venn diagram reveals minimal overlap between the three myogenic sample pairs (MB vs G₀ MB, MB vs MT, G₀ MB vs MT), and show that distinct transcriptional programs characterize reversible arrest vs. differentiation-coupled irreversible arrest. (D) Validation of microarray analysis using Northern blots. RNA samples used for microarray analysis were also interrogated with ³²P-labelled probes representing genes selected to test the range of quantitative changes in expression (i) genes that showed no change (log ratio of 0), (ii) genes that were down-regulated in G₀ (negative log ratio) or (iii) genes that were up regulated in G₀ (positive log ratio). Numbers on the left of blots represent log ratios derived from G₀ vs. asynchronous myoblast array. Gene symbols & transcript sizes are shown on the right of each blot. 28S rRNA was used as a loading control. Rfg, a gene expressed equally in all conditions. Cofilin, a regulator of microfilament dynamics - down-regulated in both G₀ Mb and in Mt. Of the 12 transcripts selected from the array as enriched in G₀, all 12 showed G0 induced expression. Of these, 8 (FGFR1, BMP1, GSTa4, TM7SF1, IGFBP5, TIMP3, Cathepsin L, Col3A1) were specifically induced in G₀ MB, and not in differentiated MT. The other 4 genes, IGF2, Rho GAP, LAMP2 and Map1LC3b were induced in both G₀ MB and MT. Histone 2B and MyoD transcripts (not derived from array analysis, marked by *) were detected to confirm the cellular state. E. Functional diversity of genes specifically enriched in G₀ MB indicates a signature of recycling (autophagy, ubiquitin pathway), resistance (anti-apoptosis) and repair (DNA repair). Gene ontology classification of 663 annotated gene from the list of genes upregulated in quiescent (G0) MB suggests that enhanced survival mechanisms may compensate for depressed metabolic functions in quiescence (see Supplementary Information for altered cell cycle and metabolic pathways). In addition, there is evidence of altered expression of genome reprogramming factors (chromatin and transcriptional regulators), signaling (growth factors, GF receptors, signaling adaptors) and developmental regulators [Wnt (indicated by arrow), Hh, BMP, Notch pathways)]. The surprising increase in induction of Wnt genes in quiescence was further investigated.

https://doi.org/10.1371/journal.pone.0065097.g002

Genes Enriched in G₀ myoblasts Define a Distinct Program of Reversible Arrest that is Overlaid by Tissue-specific Features

Hierarchical clustering (p<0.05) showed that all 3 non-cycling cellular states (G₀ MB, MT, G₀ FB) share features, but a distinct program specific to G₀ MB also emerged (Fig. 2B, C). Compared to growing myoblasts (MB), several classes of genes were discerned in the three arrested samples: (i) specifically up-regulated in G₀ MB (ii) commonly induced in G₀ MB and MT (iii) induced to a greater extent in MT than in G₀ MB (iv) commonly up-regulated in G₀ MB and G₀ FB (v) induced in G₀ FB to a greater extent than in G₀ MB.

The classic experiments of Davis et al [33] showed that forced expression of MyoD on its own could reprogram C3H10T1/2 fibroblasts to a myogenic fate. Our observations show that despite the loss of MyoD expression in myoblasts that enter G₀, the quiescence program of these closely related cell types is distinct, indicating a tissue specific layer superimposed on the common program. The overlap between the three arrested states is shown in Fig. 2C.

To independently validate array data, expression of 12 G₀-induced transcripts was confirmed by northern blot analysis (Fig. 2D). Genes selected represent a range of normalized log ratio values: NLR 0 (unchanged/control), NLR –2.01 (down-regulated in G₀) and NLR +0.99 to +3.94 (mildly to strongly up-regulated in G₀). As expected, the magnitude of differential expression varied between the two techniques (arrays vs. blot analysis), but the expression of all 12 genes tested was in concordance, validating the array analysis.

G₀-enriched Genes Define a Quiescence Program Beyond Cell Cycle Arrest

Of 1157 transcripts showing a consistent >1.6-fold induction in G₀ MB, 663 annotated genes were assigned to functional classes using gene ontology (GO) terms and pathway analysis (Fig. 2E). Suppressed proliferation (Fig. S1), and a shift to glycolysis (Table S1F), two defining features of quiescent cells were evident.

G₀-induced genes may in theory participate in programs beyond those that induce/maintain arrest per se, such as uncoupling differentiation from arrest, preserving lineage memory, activation of G₀-specific metabolic pathways, promoting survival/repair during arrest or sustaining the capacity for reactivation. Indeed, the set of genes identified as G₀-enriched were found to represent all these categories (Table S1). Here we focus on three classes of genes-tumor suppressor genes (TSGs), inhibitors of differentiation and the Wnt signaling pathway (Table 1). Induction of TSGs and inhibitors of differentiation confirm the suppression of both proliferative and tissue-specific programs in the quiescent state. However, induction of Wnt signaling in quiescent cells is surprising, as this pathway has been largely implicated in the control of proliferation [34], with additional reports suggesting a role in differentiation [35].

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Table 1. Selected classes of genes up-regulated in quiescence.

https://doi.org/10.1371/journal.pone.0065097.t001

Tumor Suppressors are Enriched in G₀

A diverse set of cell cycle inhibitors, many of which are mutated in tumors showed elevated expression in G₀. Established tumor suppressor genes induced in G₀ MB (Table 1A) include chromatin regulators (polybromo1, Klf4, PRDM2, see also Table S1G), inhibitors of transcription (p130, FOXO1, NURD complex, Meis homeodomain proteins), inhibitors of translation (Pcd4), and signaling molecules (Rab7, Dab2). These TSGs were not induced in MT, suggesting that different players inhibit the cell cycle and suppress tumorigenic potential in these two states of arrest. Interestingly, except for FoxO1, which was also induced in G₀ FB, most TSGs were specifically induced in myoblast quiescence, reinforcing the concept of a cell-type dependent quiescence program.

Suppression of proliferation is attained not only by repressing positive regulators (Cyclins, CDKs), and associated macromolecular metabolism (DNA replication, proliferation related proteins (PCNA, MCMs), RNA & protein synthesis) but also by inducing repressors such as p130, Forkhead proteins & Kruppel-like factors, known to induce quiescence in other lineages [4]. The induction of multiple TSGs suggests a strong program for failsafe inhibition of neoplastic pathways in these temporarily arrested cells.

Inhibition of Differentiation

a) Transcriptional repressors of myogenesis.

A central feature of G₀ MB is the absence of differentiation, while irreversible cell cycle exit, by contrast, is coupled to differentiation. The quiescence program includes genes that suppress entry into alternate “out of cycle” states such as differentiation [3]. Given the suppression of MyoD and lack of MyoG induction, the absence of downstream muscle genes including sarcomeric components is to be expected. However, upstream inhibitors of MyoD were also induced in G₀ indicating active suppression of determination/differentiation (Table 1B). These include repressors such as luc7-like2, Eid1, SMAD4, RBP-jk, c-jun, Stat3, Pias1, Pias 2, and HBP1, none of which were induced in MT, suggesting fail-safe pathways to protect against precocious differentiation in G₀. Interestingly, all these inhibitors of differentiation were expressed at similar levels in G₀ MB and G₀ FB except for decorin, a TGF-β pathway member. Indeed, Stat3, Pias1 and CUG-2 were enriched in G₀ FB compared to G₀ MB, reflecting the resistance of G₀ FB to MyoD-induced differentiation [2]. Induction of oncogenes such as c-jun (also seen in freshly isolated SCs [12]) lends support to the notion that the quiescence program encompasses functions beyond arrest of the cell cycle.

b) Anti-myogenic signaling network.

Figure 5. Moderate induction of Wnt signaling in two models of quiescence.

(A) Wnt signaling revealed by TCF-dependent luciferase activity in stabily transfected TOPflash myoblast clone TFC1. Asynchronously proliferating myoblasts (As), suspension-arrested quiescent myoblasts (G₀), suspension-synchronized myoblasts after re-activatation for 2–24 hrs (R2–R24), or myoblasts induced to differentiate for 72 hrs (MT). Transcriptional activity of the Wnt responsive TCF reporter is induced as MB enter quiescence and rapidly suppressed upon re-activation into the cell cycle. TCF-dependent luciferase activity (rlu/µg protein) is moderately induced in G₀ arrest but strongly induced in differentiation-associated arrest (MT, inset). Data represents mean +SE from atleast 3 independent experiments. (B) Conditioned medium (CM) from G₀ MB cultures contains more Wnt/TCF reporter-inducing activity than CM from proliferating or differentiated cultures. TOPflash reporter cells were exposed to CM derived from G₀ cultures (G₀-CM), or proliferating cultures (Mb-CM), or differentiated cultures (Mt-CM). Fresh growth medium (GM) and differentiation medium (DM) were used as controls. Data represents mean +SE from 3 independent experiments. (C) Secreted Wnt agonist R-spondin expression is strongly induced in G₀. RNA isolated from growing (Mb), arrested (G₀) and differentiated (Mt) muscle cells was analysed by Q-RT-PCR (n = 3). (D–F) Wnt signaling is induced in an independent culture model of G₀ MB (reserve cells). (D) Phase contrast photographs of quiescent mononucleated undifferentiated reserve cells (RC) after differential trypsinization specifically removed myotubes (MT) in 5-day differentiated TFC1cultures. (E) RC isolated from away from MT do not induce MyoG (QRT-PCR analysis), confirming their undifferentiated state. (F) TOPflash TCF reporter activity is induced in purified quiescent reserve cells (G₀rc) and its decline in reserve cells that have been reactivated by the addition of GM for 2–24 hrs (R2–R24). Inset shows TOPflash activity in G₀rc compared to purified MT cultures depleted of reserve cells [MT(-rc)]. Data represents mean +SE from 3 independent experiments.

https://doi.org/10.1371/journal.pone.0065097.g005

Secreted Wnt Activity is Increased in G₀

To determine whether more Wnt signaling activity is secreted in G₀, we examined the effect of conditioned media (CM) from equal numbers of MB, G₀ MB or MT derived from the parental C2C12 line, on TCF transcriptional activity in the TFC1 reporter cells. CM from MT elicited low TCF activity in reporter cells, by contrast to strong activation of TOPflash within MT. However, CM from G₀ MB elicited higher Wnt reporter activity than CM from either MB or MT (Fig. 5B).

To examine whether the disparity in secreted Wnt signals between G₀ MB and MT might be due to other secreted Wnt agonists such as R-spondin, which activate signaling via Frizzled binding [50], we used QPCR. R-spondin transcripts were induced 32-fold in G₀ but not in MT (Fig. 5C), and might contribute to Wnt pathway activation despite the elevated levels of antagonists (Frzb, sFRP4).

Intermediate, Reversible Wnt Signaling Activation in Another Quiescence Model

β-catenin, the transcriptional effector of Wnt signaling is also associated with cadherin-rich cell adhesion complexes. As detachment of cells from the substratum could cause redistribution of β-catenin, potentially increasing its nuclear availability, we considered the possibility that the suspension-induced rise of TOPflash reporter activity may reflect disruption of adhesion complexes rather than entry into G₀. To test this hypothesis, we employed an attached cell model of quiescence. Reserve cells (RC) are a minor population that arises during mitogen withdrawal-induced differentiation. These cells can constitute 10–30% of a myotube culture, and resist fusion and differentiation. Like suspension-arrested MB, RC remain mononucleated, enter G₀ and express markers of resting SCs such as Pax7 and CD34 [16]. Although not well understood, determination of RC is thought to involve Wnt and insulin signaling [51]. To further explore Wnt signaling in G₀, we generated RC from TFC1 Wnt reporter cells (Fig. 5D). MyoD (not shown) and MyoG were not expressed in RC but strongly induced in MT (Fig. 5E), validating the cell enrichment procedure. As in suspension-arrested G₀ MB, TCF activity was moderately induced in G₀ RC and down-regulated during their re-activation (Fig. 5F).

The reversible induction of TCF activity in both these quiescence models (suspension-arrested MB, attached reserve cells) suggests that Wnt signaling plays a role in G₀ irrespective of the pathway of G₀ entry, and is not merely the consequence of cell detachment. Since TCF induction is moderate in G₀ but strong in MT, either levels of Wnt activation or co-operation with different signaling pathways may distinguish G₀ from irreversible arrest. Moderate Wnt activation also appears to be important for reprogramming of somatic cells to iPS cells, while strong activation is inhibitory [52].

Taken together, these results using a transfected reporter show that the distinct and reversible Wnt expression pattern observed in quiescent myoblasts correlates with functional changes in canonical Wnt signaling that are reversed on cell cycle entry.

Differential β-catenin Occupancy at Myogenic Promoters in G₀ vs. MT

To explore the role of Wnt signaling in regulating muscle genes during reversible and irreversible arrest, we assessed occupancy of the canonical Wnt target transcription factor β-cat on endogenous myogenic promoters. Expression of Myf5, a known transcriptional target of β-cat [35], is associated with self-renewal and lineage determination and down-regulated upon overt differentiation. Myogenin (MyoG), a differentiation-inducing factor is not known to be a direct target of β-cat. Ch-IP analysis revealed that β-cat is differentially associated with Myf5 and MyoG regulatory sequences in different cellular states (Fig. 6A). In MT, where MyoG but not Myf5 is strongly expressed, β-cat was associated with chromatin at the MyoG promoter but not the Myf5 enhancer. In G₀, where neither Myf5 nor MyoG is expressed, β-cat was not found at the MyoG promoter but surprisingly, was enriched at the Myf5 enhancer. Reasoning that absence of Myf5 expression under these activating/permissive conditions might be blocked by repressive factors induced in G₀, we assessed the occupancy of a known Wnt inhibitor HBP1 (Fig. 6B). Indeed, HBP1, a repressor that directly competes with Tcf/Lef on Wnt-responsive promoters, and which was up-regulated in G₀ (Table 1), associates with the repressed Myf5 enhancer in G₀, but not with the active MyoG promoter in MT. Thus, expression of myogenic genes in G₀ vs. MT is associated with distinct combinations of Wnt-responsive transcriptional activators and repressors, and HBP1, a known myogenic and Wnt inhibitor, [53] may fine-tune regulation of muscle-specific genes in quiescence vs. differentiation.

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Figure 6. Chromatin-IP analysis of Myf5 and MyoG promoters in different cellular states.

Antibodies against β-cat or HBP were used to assess the association of these Wnt-regulated transcription factors with chromatin in different cellular states as described in Materials and Methods. Control pulldowns used IgG and all values shown represent fold enrichment of the specific transcription factor after normalization against control IgG values. (A). The Wnt target transcription factor β-cat associates with the known Wnt-responsive site in Myf5 enhancer preferentially in G₀ (G₀) but shows low enrichment in either proliferating (MB) or differentiating muscle cells (MT) (Blue bars-MB; pink bars-G₀; green bars-MT). This observation is consistent with the hypothesis that Wnt signaling is active in quiescent myoblasts. Comparison of β-cat association on another myogenic promoter (Myogenin promoter) shows greater enrichment in MT. Taken together, these observations suggest that Wnt/β-cat regulates different genes in different cellular states. (B). ChIP analysis shows that HBP1 (a Wnt-induced repressor) co-associates with the Myf5 enhancer only in G₀ (Blue bars-Mb; pink bars-G₀; green bars-MT) and does not associate with this element in either proliferating or differentiated muscle cells. This observation suggests that fine-tuning of Myf5 expression by both activating and repressive mechanisms may occur in quiescent cells by association of two types of Wnt-responsive transcription factors. Taken together, this observation would account for the absence of induction of Myf5 mRNA in quiescent myoblasts despite the association of the transcriptional activator β-cat.

https://doi.org/10.1371/journal.pone.0065097.g006

Enhancing Wnt Signaling in Suspension Culture Subverts the Quiescence Program

The results thus far indicate that reversible arrest in myoblasts is defined by a distinct transcriptional program, of which the Wnt pathway comprises a quiescence-induced module. Further, the differential elevation of TCF/β-cat transcriptional response in MT (strong) vs. G₀ (moderate) suggested that the level of Wnt signaling maybe important for achieving these distinct out-of-cycle states. Conceivably, moderate Wnt activation may be functionally linked with achieving arrest in an undifferentiated state, while strong Wnt activation may promote differentiation-coupled arrest. To test this model, we enhanced Wnt signaling in conditions that normally induce G₀, by treating suspension cultures with rWnt3a. rWnt3a was active as evidenced by induction of β-catenin translocation and TOPflash activity, both known consequences of Wnt signaling and also repressed MyoD protein (Fig. 7A). Unlike the well-documented proliferative response reported in other cell types [54] [55], rWnt3a did not activate proliferation of G₀-arrested cells (Fig. 7B). Analysis of MRF expression showed that Myf5 mRNA was induced by exogenous Wnt treatment of G₀ cells (Fig. 7C) consistent with its status as a direct Wnt target. However, MyoD and MyoG mRNAs were not induced, suggesting that strong Wnt signaling is not sufficient to re-direct G₀ towards differentiation. To test whether initial cell state is important for interpreting the Wnt signal, we added rWnt3a to proliferating, arrested or differentiated cells. Interestingly, Myf5 mRNA was induced only if target cells were already in G₀- neither MB nor MT up-regulated Myf5 in response to rWnt3a (Fig. 7D), suggesting that cellular context is important for Wnt signaling. Since many Wnt components are specifically induced in G₀, this data suggests quiescence-dependent signal responsiveness.

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Figure 7. Enhancement of Wnt signaling subverts the quiescence program.

(A) Exposure of adherent MB to rWnt3a (50 ng/ml) leads to β-cat nuclear localization, TOPflash activation and suppression of MyoD protein as compared to control cells. (B) rWnt 3a (50 ng/ml) does not enhance proliferation (BrdU incorporated in a 30′ pulse) in muscle cells: Asynchronous MB, G₀ MB, MB reactivated after synchronization in (R18) or differentiated myotubes (MT) [Note: all BrdU+ nuclei in myotube cultures were in residual mono-nucleated myobalsts]. Values represent the mean±SEM from three independent experiments. (C) Exogenous Wnt3a alters the quiescence program: Q-RTPCR analysis of control (blue bars) and Wnt-treated (pink bars) cells held in suspension for 48 hrs shows repression of MyoD and MyoG but induction of Myf5, indicating differential response of MRFs; repression of p21 and induction of CyclinD1 collectively suggesting a shift to a proliferative gene expression program; and finally, repression of quiescence-induced genes Rgs2 and Dkk3, consistent with this shift. Values represent the mean±SEM from three independent experiments. (D) Context-dependent response to Wnt enhancement. Cells in three different states (MB, G₀ or MT) were treated for 48 hours with 50ng/ml of rWnt3a. Of the MRFs, Myf5 mRNA is only induced by Wnt3a if the target cells are in G₀. Values represent the mean±SEM from three independent experiments.

https://doi.org/10.1371/journal.pone.0065097.g007

Further, β-cat (also induced in G₀) is associated with Myf5 enhancers in quiescent but not in proliferating or differentiated cells (Fig. 6), consistent with the quiescence-specific induction of Myf-5 transcript by exogenously added Wnt.

We further probed the Wnt response in G₀ by examining other genes. Firstly, the cell cycle inhibitor p21 was repressed and the pro-proliferative CyclinD1 was induced (Fig. 7C), but DNA synthesis was not activated (Fig. 7B), suggesting that while proliferation-promoting changes might be induced by Wnt3a, these were not sufficient to completely reverse arrest in non-adherent cells. Secondly, expression of two strongly quiescence-induced genes Rgs2 and Dkk3 were strongly suppressed by rWnt3a treatment. Thirdly, we employed hierarchical clustering of the QPCR “superarray” data (Table S3) to determine the degree to which Wnt treatment of G₀ myoblasts alters the Wnt network itself. rWnt3a treatment of quiescent myoblasts drastically modified the transcriptional profile of the Wnt module by suppressing quiescence-induced components (Fig. S4). Taken together, these results suggest that moderate Wnt signaling and associated Wnt component expression in G₀ may facilitate the induction/maintenance of quiescence. While elevating Wnt in G₀ subverts an actively quiescence-induced gene expression program, it is not sufficient to signal a return to active proliferation.

Strong Activation of Wnt Pathway in G₀ Reduces Clonogenic Potential

To test the functional consequences of exogenously enhanced Wnt signaling during the induction of G₀, we directly assessed cloning efficiency, a measure of self-renewal. Cells treated with 50 ng/ml rWnt3A (a dose selected on the basis of Topflash activation, Fig. S5) in adherent or suspension culture showed reduced colony formation (Fig. 8A); inclusion of Wnt inhibitor sFRP2 completely reversed this negative effect, establishing the specificity of the response to Wnt. rWnt3a treatment in G₀ did not increase either senescence (measured by senescence associated-βgal activity) nor cell death (measured by Annexin V/PI staining and FACS analysis) (Fig. S3). Thus, the self-renewal capacity associated with the modest rise in Wnt signaling normally seen during quiescence is negated by enhanced Wnt activation. Taken together, these results suggest that differences observed in the endogenous level of Wnt signaling may contribute to the distinction between reversible and irreversible arrest. Loss of clonogenicity in cells treated with Wnt may be viewed as the consequence of the altered transcriptional profile of Wnt components, which could lead to signaling conflicts that prevent cells from proliferation.

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Figure 8. Exogenous Wnt treatment compromises a G0-induced program that promotes clonogenic potential.

(A) Wnt3A treatment of MB reduces clonogenic potential. Colony formation was measured after 48 hrs in control culture conditions (either in proliferating conditions-Mb, or in suspension culture-G₀), or in the presence of 50 ng/ml of rWnt3A. Cloning efficiency (a measure of self-renewal) was strongly reduced by Wnt3A supplementation and restored by simultaneous addition of 50ng/ml sFRP2. Values represent the mean±SEM from three independent experiments, p<0.05 (denoted by asterisk *). (B) Knockdown of Rgs2 and Dkk3 transcripts using siRNAs. siRNAs were designed against the putative Wnt regulators Rgs2, Dkk3 or an irrelevant gene (GAPDH) or a control scrambled siRNA sequence and transfected into C2C12 myoblasts along with a GFP plasmid. GFP⁺ transfected cells were enriched by FACS, RNA isolated and analysed by Q-RT-PCR and the relative mRNA levels calculated. In each pair, the mRNA level is depicted of cells transfected with scrambled siRNA (blue bars) and cells transfected with the targeting siRNA (pink bars). Values represent the mean and SEM of 3 independent experiments. In each case, modest but reproducible reduction of the target transcript level is observed. (C) Reduction of Rgs2 and Dkk3 protein expression by siRNA-mediated knockdown. Western blot analysis of total protein isolated from control and knockdown C2C12 muscle cells probed with antibodies against Rgs2 (top) and Dkk3 (bottom). GAPDH protein levels indicate equal loading. Data depicted is representative of 3 independent experiments. (D) Rgs2 and Dkk3 expression is necessary for Wnt signaling. Knockdown of either Rgs2 or Dkk3 in growing or quiescent MB leads to suppression of TOPflash activity. Cells were treated and enriched as described in (B) and luciferase activity measured. Despite modest reduction of protein levels, strong reduction in TOPflash activity are seen, indicating a critical role for Rgs2 and Dkk3 in Wnt-βcat signaling. Values represent the mean and SEM of 3 independent experiments. (E,F) Knockdown cells (‘Rgs sh’ and ‘Dkk sh’) were enriched as described in (B) cultured in quiescence-inducing conditions, recovered from suspension culture and plated at clonogenic density for assessment of self-renewal (colony formation). Controls include untransfected cells (‘UT’) and control shRNA transfected cells (‘Con sh’). Typical plates with colony assays are shown in (E) and data are quantified as CFU (colony forming units) in (F). Values represent the mean and SEM of 3 independent experiments.

https://doi.org/10.1371/journal.pone.0065097.g008

Quiescence-induced Genes RGS2 and DKK3 are Required for Wnt Signaling and Promote Self-renewal

The strong induction of Rgs2 and Dkk3 specifically in G₀ MB but not MT, and their rapid suppression as G₀ cells entered G₁ (Fig. 4E) is consistent with a role in the quiescence program. Expression of both Rgs2 and Dkk3 was suppressed when quiescent myoblasts were exposed to rWnt3a (Fig. 7C), coincident with loss of clonogenicity, and suggests a role in self-renewal. Rgs2 regulates heterotrimeric G protein (Gα) signaling in cardiac cells, but in Xenopus, ectopically expressed hRgs2 phenocopies dominant-negative XWnt8 [46], suggesting Wnt inhibitory functions. Dkk3 is related to known Wnt antagonist Dkk1, but is not thought to inhibit Wnt [48], and may even promote Wnt signaling [47]. However, the function of these genes in myoblasts was unknown. To test Rgs2 and Dkk3 function in quiescent MB, we assessed their potential role in canonical Wnt signaling. Knockdown of either Rgs2 or Dkk3 at RNA and protein level (Fig. 8B,C) suppressed canonical Wnt signaling, as evidenced by reduced TOPflash activity (Fig. 8D). Thus, in quiescent MB, rather than inhibiting the Wnt pathway, both Rgs2 and Dkk3 appear to be required for Wnt signaling.

Artificially elevated Wnt signaling in G₀ led to loss of Rgs2/Dkk3 expression as well as reduced cloning efficiency. To investigate a possible causal relationship between Rgs2/Dkk3 expression and clonogenic potential, we used RNAi. Myoblasts in which either Rgs2 or Dkk3 were knocked down were FACS-enriched (using a co-transfected GFP plasmid), cultured in suspension for 48 h to induce G₀, and analyzed by CFU assay. Colony formation in both Rgs2 and Dkk3 knockdown cells was reduced compared to control transfected or untransfected cells (Fig. 8E,F), supporting the hypothesis that these quiescence-induced, Wnt regulatory genes play a critical role in maintaining self-renewal potential in G₀.

Collectively, these studies demonstrate that the Wnt signature identified as enriched in G₀ myoblasts by unbiased profiling has functional consequences for quiescence, and identify two new regulators of the quiescence program, Rgs2 and Dkk3, both of which are required for canonical Wnt signaling in quiescent cells.

Summary and Conclusions

The control of reversible quiescence, a cellular state with important implications for stem cell function and tumor biology is incompletely defined. A core quiescence program has been described in lymphocytes, HSC and fibroblasts [2] [4] [5], but no genome-wide analysis has compared reversible arrest to other physiological, viable out-of-cycle states. Several important concepts emerge from our analysis. Firstly, distinct genetic programs control reversible and irreversible [differentiation-associated] arrest. A key component of the reversible quiescence program in myoblasts is an active suppression of differentiation by multiple failsafe mechanisms. This muscle inhibitory program is also induced in G₀ fibroblasts, and could account for their ability to resist differentiation by ectopic MyoD [2] [3]. Secondly, tissue-specific features overlay the core quiescence program. Although G₀ MB and G₀ FB share a common transcriptional profile, a significant number of unique genes are enriched in each cell type alone. Thirdly, a distinct constellation of tumor suppressors/cell cycle inhibitors is induced in reversible arrest, which in combination with the large number of inhibitors of differentiation emphasizes distinct strategies for achieving and maintaining the quiescent state. Fourthly, a number of genes are conserved between cultured G₀ MB and freshly isolated SC, which may reflect conserved strategies for survival/self-renewal. Finally, Wnt signaling, a key regulator of stem cell self renewal regulates the induction/maintenance of the quiescent state. The surprising association of Wnt signaling with reversible arrest is discussed in detail below.

Unexpected Association of Wnt Signaling with the Quiescent State

Wnt signaling is most commonly associated with proliferation and its deregulation is clearly implicated in tumorigenesis [44]. However, growing evidence suggests context- and cell type-dependent interpretation of Wnt signals [44], [56]with outcomes other than proliferation (such as differentiation, apoptosis [43]). In addition to the intrinsic complexity of the Wnt pathway (multiple Wnt ligands, receptors, co-receptors, co-inhibitors and target transcription factors), there are synergistic and antagonistic interactions with other signaling pathways. Viewed against this complexity, the distinct state-specific outcomes we report in the myogenic cell system provide an opportunity to distinguish context-dependent sub-networks.

Three observations support the view that the distinct Wnt pathway transcriptional signature in quiescent MB reflects functional Wnt signaling. First, canonical Wnt pathway reporter activity showed a moderate rise in G₀, reaching a level intermediate between levels in proliferating and differentiated muscle cells. The intermediate level appears to be important since enhancing Wnt levels in G₀ myoblasts by addition of exogenous rWnt3a protein not only altered expression patterns typical of G₀ myoblasts [myogenic factors, quiescence-induced genes, cell cycle genes, Wnt genes], but also had deleterious effects on clonogenicity. These observations suggest that signaling through the endogenous Wnt pathway is regulated and moderate induction provides a specific benefit to quiescent cells. Second, two genes most highly induced in G₀ (Rgs2 and Dkk3) were found to be required for canonical Wnt signaling. Third, altering expression of these Wnt pathway activators (Rgs2 and Dkk3) negatively affected clonogenic survival of G₀ MB. Taken together, these results strongly support a functional role for moderate activation of the Wnt pathway in attaining/maintaining quiescence. This leads us to speculate that a threshold level of Wnt signaling may be essential for survival in G₀, but over-shooting that level is restrictive.

Wnt Signaling, Survival and Self-renewal

The context-dependent role of Wnt in muscle cells also suggests that other factors (such as IGFs) may co-ordinately induce differentiation. Wnt and insulin have synergistic effects on muscle differentiation and hypertrophy [51], and in G₀ MB, lower expression of IGFs, IGFR and IGF-BP than in MT may contribute to their undifferentiated state. Loss of MyoD expression when nuclear β-cat expression is induced would also reinforce the undifferentiated state.

An important function of Wnt signaling may be to promote cell survival [54], through IGFs, [57] and PI3 kinase/Akt [58] [59]. Thus, in G₀ MB, moderate Wnt activation and low levels of IGFs may combine to protect cells against apoptosis and aid in cell survival by activating the PI3kinase/Akt pathway. Our finding that colony formation is adversely affected when Wnt signaling is enhanced supports this notion.

We thank D. Bowtell, P. Bresnahan, S. Pickett, B. Ramachandran, M. van de Rijn, Suchitra Gopinath, T. Vaidya for helpful discussions; Surabhi Srivastava for help with bioinformatic analysis, G. Nagendra and Asha Ramesh for technical support; A. Daramarajam, C. Niehrs, M. Waterman and R. Moon for generous gifts of reagents; Apurva Sarin and R. Sambasivan for critical reading of the manuscript.

Author Contributions

Conceived and designed the experiments: SS PS JD. Performed the experiments: SS PS SC MM. Analyzed the data: SS PS SC VRR MM RKC LSS JD. Contributed reagents/materials/analysis tools: LSS. Wrote the paper: SS PS JD.

References

1. Dhawan J, Rando TA (2005) Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol 15: 666–673.
- View Article
- Google Scholar
2. Coller HA, Sang L, Roberts JM (2006) A new description of cellular quiescence. PLoS Biol 4: e83.
- View Article
- Google Scholar
3. Sang L, Coller HA, Roberts JM (2008) Control of the reversibility of cellular quiescence by the transcriptional repressor HES1. Science 321: 1095–1100.
- View Article
- Google Scholar
4. Yusuf I, Fruman DA (2003) Regulation of quiescence in lymphocytes. Trends Immunol 24: 380–386.
- View Article
- Google Scholar
5. Venezia TA, Merchant AA, Ramos CA, Whitehouse NL, Young AS, et al. (2004) Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol 2: e301.
- View Article
- Google Scholar
6. Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, et al. (2000) Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 287: 1804–1808.
- View Article
- Google Scholar
7. Nurse P (2000) The incredible life and times of biological cells. Science 289: 1711–1716.
- View Article
- Google Scholar
8. Gray JV, Petsko GA, Johnston GC, Ringe D, Singer RA, et al. (2004) “Sleeping beauty”: quiescence in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 68: 187–206.
- View Article
- Google Scholar
9. Yanagida M (2009) Cellular quiescence: are controlling genes conserved? Trends Cell Biol 19: 705–715.
- View Article
- Google Scholar
10. Liu H, Adler AS, Segal E, Chang HY (2007) A transcriptional program mediating entry into cellular quiescence. PLoS Genet 3: e91.
- View Article
- Google Scholar
11. Sebastian S, Sreenivas P, Sambasivan R, Cheedipudi S, Kandalla P, et al. (2009) MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation. Proc Natl Acad Sci U S A 106: 4719–4724.
- View Article
- Google Scholar
12. Fukada S, Uezumi A, Ikemoto M, Masuda S, Segawa M, et al. (2007) Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25: 2448–2459.
- View Article
- Google Scholar
13. Pallafacchina G, Francois S, Regnault B, Czarny B, Dive V, et al. (2010) An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells. Stem Cell Res 4: 77–91.
- View Article
- Google Scholar
14. Andres V, Walsh K (1996) Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J Cell Biol 132: 657–666.
- View Article
- Google Scholar
15. Blau HM, Webster C, Chiu CP, Guttman S, Chandler F (1983) Differentiation properties of pure populations of human dystrophic muscle cells. Exp Cell Res 144: 495–503.
- View Article
- Google Scholar
16. Yoshida N, Yoshida S, Koishi K, Masuda K, Nabeshima Y (1998) Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’. J Cell Sci 111 (Pt 6): 769–779.
- View Article
- Google Scholar
17. Milasincic DJ, Dhawan J, Farmer SR (1996) Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts. In Vitro Cell Dev Biol Anim 32: 90–99.
- View Article
- Google Scholar
18. Sachidanandan C, Sambasivan R, Dhawan J (2002) Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury. J Cell Sci 115: 2701–2712.
- View Article
- Google Scholar
19. Beauchamp JR, Heslop L, Yu DS, Tajbakhsh S, Kelly RG, et al. (2000) Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 151: 1221–1234.
- View Article
- Google Scholar
20. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, et al. (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102: 777–786.
- View Article
- Google Scholar
21. Cornelison DD, Wold BJ (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191: 270–283.
- View Article
- Google Scholar
22. Sambasivan R, Cheedipudi S, Pasupuleti N, Saleh A, Pavlath GK, et al. (2009) The small chromatin-binding protein p8 coordinates the association of anti-proliferative and pro-myogenic proteins at the myogenin promoter. J Cell Sci 122: 3481–3491.
- View Article
- Google Scholar
23. Yaffe D, Saxel O (1977) A myogenic cell line with altered serum requirements for differentiation. Differentiation 7: 159–166.
- View Article
- Google Scholar
24. Kitzmann M, Carnac G, Vandromme M, Primig M, Lamb NJ, et al. (1998) The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol 142: 1447–1459.
- View Article
- Google Scholar
25. Gopinath SD, Narumiya S, Dhawan J (2007) The RhoA effector mDiaphanous regulates MyoD expression and cell cycle progression via SRF-dependent and SRF-independent pathways. J Cell Sci 120: 3086–3098.
- View Article
- Google Scholar
26. Ko MS, Threat TA, Wang X, Horton JH, Cui Y, et al. (1998) Genome-wide mapping of unselected transcripts from extraembryonic tissue of 7.5-day mouse embryos reveals enrichment in the t-complex and under-representation on the X chromosome. Hum Mol Genet 7: 1967–1978.
- View Article
- Google Scholar
27. Edmondson DG, Cheng TC, Cserjesi P, Chakraborty T, Olson EN (1992) Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2. Mol Cell Biol 12: 3665–3677.
- View Article
- Google Scholar
28. Buchberger A, Nomokonova N, Arnold HH (2003) Myf5 expression in somites and limb buds of mouse embryos is controlled by two distinct distal enhancer activities. Development 130: 3297–3307.
- View Article
- Google Scholar
29. Walsh K, Perlman H (1997) Cell cycle exit upon myogenic differentiation. Curr Opin Genet Dev 7: 597–602.
- View Article
- Google Scholar
30. Dhawan J, Helfman DM (2004) Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation. J Cell Sci 117: 3735–3748.
- View Article
- Google Scholar
31. Clegg CH, Linkhart TA, Olwin BB, Hauschka SD (1987) Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J Cell Biol 105: 949–956.
- View Article
- Google Scholar
32. Florini JR, Ewton DZ, Coolican SA (1996) Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17: 481–517.
- View Article
- Google Scholar
33. Davis RL, Weintraub H, Lassar AB (1987) Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987–1000.
- View Article
- Google Scholar
34. Otto A, Schmidt C, Luke G, Allen S, Valasek P, et al. (2008) Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. J Cell Sci 121: 2939–2950.
- View Article
- Google Scholar
35. Borello U, Berarducci B, Murphy P, Bajard L, Buffa V, et al. (2006) The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis. Development 133: 3723–3732.
- View Article
- Google Scholar
36. Coffey RJ Jr, Bascom CC, Sipes NJ, Graves-Deal R, Weissman BE, et al. (1988) Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta. Mol Cell Biol 8: 3088–3093.
- View Article
- Google Scholar
37. Serra R, Moses HL (1996) Tumor suppressor genes in the TGF-beta signaling pathway? Nat Med 2: 390–391.
- View Article
- Google Scholar
38. Luo K (2004) Ski and SnoN: negative regulators of TGF-beta signaling. Curr Opin Genet Dev 14: 65–70.
- View Article
- Google Scholar
39. Nusse R (2008) Wnt signaling and stem cell control. Cell Res 18: 523–527.
- View Article
- Google Scholar
40. Alonso L, Fuchs E (2003) Stem cells in the skin: waste not, Wnt not. Genes Dev 17: 1189–1200.
- View Article
- Google Scholar
41. Tajbakhsh S, Borello U, Vivarelli E, Kelly R, Papkoff J, et al. (1998) Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development 125: 4155–4162.
- View Article
- Google Scholar
42. Cossu G, Borello U (1999) Wnt signaling and the activation of myogenesis in mammals. EMBO J 18: 6867–6872.
- View Article
- Google Scholar
43. Brack AS, Conboy MJ, Roy S, Lee M, Kuo CJ, et al. (2007) Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science 317: 807–810.
- View Article
- Google Scholar
44. Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell 127: 469–480.
- View Article
- Google Scholar
45. Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, et al. (2003) A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423: 409–414.
- View Article
- Google Scholar
46. Wu C, Zeng Q, Blumer KJ, Muslin AJ (2000) RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development. Development 127: 2773–2784.
- View Article
- Google Scholar
47. Nakamura RE, Hunter DD, Yi H, Brunken WJ, Hackam AS (2007) Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina. BMC Cell Biol 8: 52.
- View Article
- Google Scholar
48. Niehrs C (2006) Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25: 7469–7481.
- View Article
- Google Scholar
49. Veeman MT, Slusarski DC, Kaykas A, Louie SH, Moon RT (2003) Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr Biol 13: 680–685.
- View Article
- Google Scholar
50. Kazanskaya O, Glinka A, del Barco Barrantes I, Stannek P, Niehrs C, et al. (2004) R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev Cell 7: 525–534.
- View Article
- Google Scholar
51. Rochat A, Fernandez A, Vandromme M, Moles JP, Bouschet T, et al. (2004) Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy. Mol Biol Cell 15: 4544–4555.
- View Article
- Google Scholar
52. Lluis F, Pedone E, Pepe S, Cosma MP (2008) Periodic activation of Wnt/beta-catenin signaling enhances somatic cell reprogramming mediated by cell fusion. Cell Stem Cell 3: 493–507.
- View Article
- Google Scholar
53. Sampson EM, Haque ZK, Ku MC, Tevosian SG, Albanese C, et al. (2001) Negative regulation of the Wnt-beta-catenin pathway by the transcriptional repressor HBP1. EMBO J 20: 4500–4511.
- View Article
- Google Scholar
54. Dravid G, Ye Z, Hammond H, Chen G, Pyle A, et al. (2005) Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells 23: 1489–1501.
- View Article
- Google Scholar
55. Fleming HE, Janzen V, Lo Celso C, Guo J, Leahy KM, et al. (2008) Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell Stem Cell 2: 274–283.
- View Article
- Google Scholar
56. Nalesso G, Sherwood J, Bertrand J, Pap T, Ramachandran M, et al. (2011) WNT-3A modulates articular chondrocyte phenotype by activating both canonical and noncanonical pathways. J Cell Biol 193: 551–564.
- View Article
- Google Scholar
57. Longo KA, Kennell JA, Ochocinska MJ, Ross SE, Wright WS, et al. (2002) Wnt signaling protects 3T3-L1 preadipocytes from apoptosis through induction of insulin-like growth factors. J Biol Chem 277: 38239–38244.
- View Article
- Google Scholar
58. Almeida M, Han L, Bellido T, Manolagas SC, Kousteni S (2005) Wnt proteins prevent apoptosis of both uncommitted osteoblast progenitors and differentiated osteoblasts by beta-catenin-dependent and -independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase/AKT. J Biol Chem 280: 41342–41351.
- View Article
- Google Scholar
59. Sinha D, Wang Z, Ruchalski KL, Levine JS, Krishnan S, et al. (2005) Lithium activates the Wnt and phosphatidylinositol 3-kinase Akt signaling pathways to promote cell survival in the absence of soluble survival factors. Am J Physiol Renal Physiol 288: F703–713.
- View Article
- Google Scholar
60. Molofsky AV, Pardal R, Morrison SJ (2004) Diverse mechanisms regulate stem cell self-renewal. Curr Opin Cell Biol 16: 700–707.
- View Article
- Google Scholar

[ref1] 1. Dhawan J, Rando TA (2005) Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol 15: 666–673.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Coller HA, Sang L, Roberts JM (2006) A new description of cellular quiescence. PLoS Biol 4: e83.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Sang L, Coller HA, Roberts JM (2008) Control of the reversibility of cellular quiescence by the transcriptional repressor HES1. Science 321: 1095–1100.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. Yusuf I, Fruman DA (2003) Regulation of quiescence in lymphocytes. Trends Immunol 24: 380–386.
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref5] 5. Venezia TA, Merchant AA, Ramos CA, Whitehouse NL, Young AS, et al. (2004) Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol 2: e301.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref6] 6. Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, et al. (2000) Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 287: 1804–1808.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref7] 7. Nurse P (2000) The incredible life and times of biological cells. Science 289: 1711–1716.
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref8] 8. Gray JV, Petsko GA, Johnston GC, Ringe D, Singer RA, et al. (2004) “Sleeping beauty”: quiescence in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 68: 187–206.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref9] 9. Yanagida M (2009) Cellular quiescence: are controlling genes conserved? Trends Cell Biol 19: 705–715.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref10] 10. Liu H, Adler AS, Segal E, Chang HY (2007) A transcriptional program mediating entry into cellular quiescence. PLoS Genet 3: e91.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref11] 11. Sebastian S, Sreenivas P, Sambasivan R, Cheedipudi S, Kandalla P, et al. (2009) MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation. Proc Natl Acad Sci U S A 106: 4719–4724.
View Article
Google Scholar

[32] View Article

[33] Google Scholar

[ref12] 12. Fukada S, Uezumi A, Ikemoto M, Masuda S, Segawa M, et al. (2007) Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25: 2448–2459.
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref13] 13. Pallafacchina G, Francois S, Regnault B, Czarny B, Dive V, et al. (2010) An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells. Stem Cell Res 4: 77–91.
View Article
Google Scholar

[38] View Article

[39] Google Scholar

[ref14] 14. Andres V, Walsh K (1996) Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J Cell Biol 132: 657–666.
View Article
Google Scholar

[41] View Article

[42] Google Scholar

[ref15] 15. Blau HM, Webster C, Chiu CP, Guttman S, Chandler F (1983) Differentiation properties of pure populations of human dystrophic muscle cells. Exp Cell Res 144: 495–503.
View Article
Google Scholar

[44] View Article

[45] Google Scholar

[ref16] 16. Yoshida N, Yoshida S, Koishi K, Masuda K, Nabeshima Y (1998) Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’. J Cell Sci 111 (Pt 6): 769–779.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref17] 17. Milasincic DJ, Dhawan J, Farmer SR (1996) Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts. In Vitro Cell Dev Biol Anim 32: 90–99.
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref18] 18. Sachidanandan C, Sambasivan R, Dhawan J (2002) Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury. J Cell Sci 115: 2701–2712.
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref19] 19. Beauchamp JR, Heslop L, Yu DS, Tajbakhsh S, Kelly RG, et al. (2000) Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 151: 1221–1234.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref20] 20. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, et al. (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102: 777–786.
View Article
Google Scholar

[59] View Article

[60] Google Scholar

[ref21] 21. Cornelison DD, Wold BJ (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191: 270–283.
View Article
Google Scholar

[62] View Article

[63] Google Scholar

[ref22] 22. Sambasivan R, Cheedipudi S, Pasupuleti N, Saleh A, Pavlath GK, et al. (2009) The small chromatin-binding protein p8 coordinates the association of anti-proliferative and pro-myogenic proteins at the myogenin promoter. J Cell Sci 122: 3481–3491.
View Article
Google Scholar

[65] View Article

[66] Google Scholar

[ref23] 23. Yaffe D, Saxel O (1977) A myogenic cell line with altered serum requirements for differentiation. Differentiation 7: 159–166.
View Article
Google Scholar

[68] View Article

[69] Google Scholar

[ref24] 24. Kitzmann M, Carnac G, Vandromme M, Primig M, Lamb NJ, et al. (1998) The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol 142: 1447–1459.
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref25] 25. Gopinath SD, Narumiya S, Dhawan J (2007) The RhoA effector mDiaphanous regulates MyoD expression and cell cycle progression via SRF-dependent and SRF-independent pathways. J Cell Sci 120: 3086–3098.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref26] 26. Ko MS, Threat TA, Wang X, Horton JH, Cui Y, et al. (1998) Genome-wide mapping of unselected transcripts from extraembryonic tissue of 7.5-day mouse embryos reveals enrichment in the t-complex and under-representation on the X chromosome. Hum Mol Genet 7: 1967–1978.
View Article
Google Scholar

[77] View Article

[78] Google Scholar

[ref27] 27. Edmondson DG, Cheng TC, Cserjesi P, Chakraborty T, Olson EN (1992) Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2. Mol Cell Biol 12: 3665–3677.
View Article
Google Scholar

[80] View Article

[81] Google Scholar

[ref28] 28. Buchberger A, Nomokonova N, Arnold HH (2003) Myf5 expression in somites and limb buds of mouse embryos is controlled by two distinct distal enhancer activities. Development 130: 3297–3307.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref29] 29. Walsh K, Perlman H (1997) Cell cycle exit upon myogenic differentiation. Curr Opin Genet Dev 7: 597–602.
View Article
Google Scholar

[86] View Article

[87] Google Scholar

[ref30] 30. Dhawan J, Helfman DM (2004) Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation. J Cell Sci 117: 3735–3748.
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref31] 31. Clegg CH, Linkhart TA, Olwin BB, Hauschka SD (1987) Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J Cell Biol 105: 949–956.
View Article
Google Scholar

[92] View Article

[93] Google Scholar

[ref32] 32. Florini JR, Ewton DZ, Coolican SA (1996) Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17: 481–517.
View Article
Google Scholar

[95] View Article

[96] Google Scholar

[ref33] 33. Davis RL, Weintraub H, Lassar AB (1987) Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987–1000.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref34] 34. Otto A, Schmidt C, Luke G, Allen S, Valasek P, et al. (2008) Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. J Cell Sci 121: 2939–2950.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref35] 35. Borello U, Berarducci B, Murphy P, Bajard L, Buffa V, et al. (2006) The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis. Development 133: 3723–3732.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref36] 36. Coffey RJ Jr, Bascom CC, Sipes NJ, Graves-Deal R, Weissman BE, et al. (1988) Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta. Mol Cell Biol 8: 3088–3093.
View Article
Google Scholar

[107] View Article

[108] Google Scholar

[ref37] 37. Serra R, Moses HL (1996) Tumor suppressor genes in the TGF-beta signaling pathway? Nat Med 2: 390–391.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref38] 38. Luo K (2004) Ski and SnoN: negative regulators of TGF-beta signaling. Curr Opin Genet Dev 14: 65–70.
View Article
Google Scholar

[113] View Article

[114] Google Scholar

[ref39] 39. Nusse R (2008) Wnt signaling and stem cell control. Cell Res 18: 523–527.
View Article
Google Scholar

[116] View Article

[117] Google Scholar

[ref40] 40. Alonso L, Fuchs E (2003) Stem cells in the skin: waste not, Wnt not. Genes Dev 17: 1189–1200.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref41] 41. Tajbakhsh S, Borello U, Vivarelli E, Kelly R, Papkoff J, et al. (1998) Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development 125: 4155–4162.
View Article
Google Scholar

[122] View Article

[123] Google Scholar

[ref42] 42. Cossu G, Borello U (1999) Wnt signaling and the activation of myogenesis in mammals. EMBO J 18: 6867–6872.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref43] 43. Brack AS, Conboy MJ, Roy S, Lee M, Kuo CJ, et al. (2007) Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science 317: 807–810.
View Article
Google Scholar

[128] View Article

[129] Google Scholar

[ref44] 44. Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell 127: 469–480.
View Article
Google Scholar

[131] View Article

[132] Google Scholar

[ref45] 45. Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, et al. (2003) A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423: 409–414.
View Article
Google Scholar

[134] View Article

[135] Google Scholar

[ref46] 46. Wu C, Zeng Q, Blumer KJ, Muslin AJ (2000) RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development. Development 127: 2773–2784.
View Article
Google Scholar

[137] View Article

[138] Google Scholar

[ref47] 47. Nakamura RE, Hunter DD, Yi H, Brunken WJ, Hackam AS (2007) Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina. BMC Cell Biol 8: 52.
View Article
Google Scholar

[140] View Article

[141] Google Scholar

[ref48] 48. Niehrs C (2006) Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25: 7469–7481.
View Article
Google Scholar

[143] View Article

[144] Google Scholar

[ref49] 49. Veeman MT, Slusarski DC, Kaykas A, Louie SH, Moon RT (2003) Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr Biol 13: 680–685.
View Article
Google Scholar

[146] View Article

[147] Google Scholar

[ref50] 50. Kazanskaya O, Glinka A, del Barco Barrantes I, Stannek P, Niehrs C, et al. (2004) R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev Cell 7: 525–534.
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref51] 51. Rochat A, Fernandez A, Vandromme M, Moles JP, Bouschet T, et al. (2004) Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy. Mol Biol Cell 15: 4544–4555.
View Article
Google Scholar

[152] View Article

[153] Google Scholar

[ref52] 52. Lluis F, Pedone E, Pepe S, Cosma MP (2008) Periodic activation of Wnt/beta-catenin signaling enhances somatic cell reprogramming mediated by cell fusion. Cell Stem Cell 3: 493–507.
View Article
Google Scholar

[155] View Article

[156] Google Scholar

[ref53] 53. Sampson EM, Haque ZK, Ku MC, Tevosian SG, Albanese C, et al. (2001) Negative regulation of the Wnt-beta-catenin pathway by the transcriptional repressor HBP1. EMBO J 20: 4500–4511.
View Article
Google Scholar

[158] View Article

[159] Google Scholar

[ref54] 54. Dravid G, Ye Z, Hammond H, Chen G, Pyle A, et al. (2005) Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells 23: 1489–1501.
View Article
Google Scholar

[161] View Article

[162] Google Scholar

[ref55] 55. Fleming HE, Janzen V, Lo Celso C, Guo J, Leahy KM, et al. (2008) Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell Stem Cell 2: 274–283.
View Article
Google Scholar

[164] View Article

[165] Google Scholar

[ref56] 56. Nalesso G, Sherwood J, Bertrand J, Pap T, Ramachandran M, et al. (2011) WNT-3A modulates articular chondrocyte phenotype by activating both canonical and noncanonical pathways. J Cell Biol 193: 551–564.
View Article
Google Scholar

[167] View Article

[168] Google Scholar

[ref57] 57. Longo KA, Kennell JA, Ochocinska MJ, Ross SE, Wright WS, et al. (2002) Wnt signaling protects 3T3-L1 preadipocytes from apoptosis through induction of insulin-like growth factors. J Biol Chem 277: 38239–38244.
View Article
Google Scholar

[170] View Article

[171] Google Scholar

[ref58] 58. Almeida M, Han L, Bellido T, Manolagas SC, Kousteni S (2005) Wnt proteins prevent apoptosis of both uncommitted osteoblast progenitors and differentiated osteoblasts by beta-catenin-dependent and -independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase/AKT. J Biol Chem 280: 41342–41351.
View Article
Google Scholar

[173] View Article

[174] Google Scholar

[ref59] 59. Sinha D, Wang Z, Ruchalski KL, Levine JS, Krishnan S, et al. (2005) Lithium activates the Wnt and phosphatidylinositol 3-kinase Akt signaling pathways to promote cell survival in the absence of soluble survival factors. Am J Physiol Renal Physiol 288: F703–713.
View Article
Google Scholar

[176] View Article

[177] Google Scholar

[ref60] 60. Molofsky AV, Pardal R, Morrison SJ (2004) Diverse mechanisms regulate stem cell self-renewal. Curr Opin Cell Biol 16: 700–707.
View Article
Google Scholar

[179] View Article

[180] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Cell Culture

Transfection

Immunofluorescence

Microarray Analysis

Hybridization.

Array informatics and statistics.

Cell cycle analysis.

Wnt superarray.

Knockdown analysis.

Chromatin Immuno-Precipitation (ChIP) assay.

Colony assays (CFU).

Results and Discussion

Model System

Microarray Profiling of Arrested Myoblasts and Fibroblasts: Experimental Design and Data Analysis

Genes Enriched in G0 myoblasts Define a Distinct Program of Reversible Arrest that is Overlaid by Tissue-specific Features

G0-enriched Genes Define a Quiescence Program Beyond Cell Cycle Arrest

Tumor Suppressors are Enriched in G0

Inhibition of Differentiation

a) Transcriptional repressors of myogenesis.

b) Anti-myogenic signaling network.

Genes Enriched in Quiescent Muscle Stem Cells in vivo are Induced during G0 in Culture

Developmental Signaling Pathways and Quiescence –a Wnt Signaling Signature

Validation of Wnt Pathway Gene Expression in G0

Functional Analysis of Canonical Wnt Signaling Reveals differences between G0 Myoblasts and Myotubes

Secreted Wnt Activity is Increased in G0

Intermediate, Reversible Wnt Signaling Activation in Another Quiescence Model

Differential β-catenin Occupancy at Myogenic Promoters in G0 vs. MT

Enhancing Wnt Signaling in Suspension Culture Subverts the Quiescence Program

Strong Activation of Wnt Pathway in G0 Reduces Clonogenic Potential

Quiescence-induced Genes RGS2 and DKK3 are Required for Wnt Signaling and Promote Self-renewal

Summary and Conclusions

Unexpected Association of Wnt Signaling with the Quiescent State

Wnt Signaling, Survival and Self-renewal

Supporting Information

Acknowledgments

Author Contributions

References

Genes Enriched in G₀ myoblasts Define a Distinct Program of Reversible Arrest that is Overlaid by Tissue-specific Features

G₀-enriched Genes Define a Quiescence Program Beyond Cell Cycle Arrest

Tumor Suppressors are Enriched in G₀

Genes Enriched in Quiescent Muscle Stem Cells in vivo are Induced during G₀ in Culture

Validation of Wnt Pathway Gene Expression in G₀

Functional Analysis of Canonical Wnt Signaling Reveals differences between G₀ Myoblasts and Myotubes

Secreted Wnt Activity is Increased in G₀

Differential β-catenin Occupancy at Myogenic Promoters in G₀ vs. MT

Strong Activation of Wnt Pathway in G₀ Reduces Clonogenic Potential