Expression data from Rspo1-null differentiating primary myoblasts
ABSTRACT: Regeneration of the adult skeletal muscle tissue relies on a population of muscle stem cells called satellite cells. During tisse repair, satellite cells exhibit active canonical Wnt/beta-catenin signaling. Rspo1 is a modulator of Wnt signaling in many tissue, and is expressed by muscle progenitor cells. To identify genes that become transcribed following activation of the Wnt/β-Catenin pathway in satellite cells, we performed microarray analysis of primary myoblasts expressing an active form of beta-catenin Overall design: Primary myoblasts were prepared from the limb muscles of wild-type and Rpos1-null mice. RNA was extracted from two independent cultures for each conditions.
INSTRUMENT(S): [MoGene-2_0-st] Affymetrix Mouse Gene 2.0 ST Array (mogene20st_Mm_ENTREZG_19)
Project description:Regeneration of the adult skeletal muscle tissue relies on a population of muscle stem cells called satellite cells. During tissue repair, satellite cells exhibit active canonical Wnt/beta-catenin signaling. To identify genes that become transcribed following activation of the Wnt/β-Catenin pathway in satellite cells, we performed microarray analysis of primary myoblasts expressing an active form of beta-catenin Overall design: Primary myoblasts were prepared from the limb muscles of Pax7-CreERT2*Beta-cetenin-Exon3-loxP/+ mice. Conditional activation of the beta-catenin gene was validated by genotyping PCR and Western Blotting. RNA was extracted from three independent cultures for each conditions.
Project description:APC is a key regulator of canonical Wnt signalling since it participates to beta-catenin targeting to proteasomal degradation when the pathway is inactive. Moreover, independently of Wnt signaling, APC regulates several cellular functions such as mycrotubule dynamics, chromosome segregation, cell adhesion. Although APC has been widely studied for its implication in initation and progression of several cancers, its role in satellite cells (skeletal muscle stem cells) has never been investigated. Here we used microarrays and to clarify APC functions and we identified several pathways and cellular processes to be affected following APC silencing. Overall design: We cultured satellite cells-derived primary myoblasts in proliferating conditions and we transfected them either by a siRNA targeting APC or by a control siRNA. 48 hours after transfection, cells were processed for RNA extraction.
Project description:Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However, both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown. Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation. These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation. Control cells (day 0) prior to differentiation induction with n=4; differentiated for two days with n=3; differentiated for four days with n=3.
Project description:Satellite cells play an important role in post-natal growth and regeneration of skeletal muscle. They can be defined as a population adult muscle stem cells based on their self renewal capability and ability to differentiate into skeletal muscle fibers. Functional Retinoblastoma protein (pRb) is essential for the process of skeletal muscle differentiation in satellite cell derived primary myoblasts. Furthermore, the biochemical function of pRb is largely associated with its ability to interact with chromatin modifying factors such as histone deacetylases (HDACs) and histone methyltransferases thus inhibiting transcription of target gene promoters. Hence, expression profiling of pRb null primary myoblasts and myotubes will provide a global picture of the downstream targets of pRb transcriptional regulation in relation to cell cycle control, apoptosis inhibition, and muscle differentiation. Keywords: other Overall design: This experiment includes 4 samples with 3 replicates each on 2 platforms for a total of 24 Samples.
Project description:Muscle satellite cells are a self-renewing pool of stem cells that give rise to daughter myogenic precursor cells in adult skeletal muscle. Published and preliminary data indicated that MyoD and p53 genes are involved in satellite cell differentiation. We would like to know what downstream genes of both transcription factors are affected in satellite cell-derived myoblasts (MyoD-/-, p53 -/-). Experiment Overall Design: this experiment include 3 samples and 25 replicates
Project description:The canonical Wnt signaling pathway is critical for myogenesis and can induce muscle progenitors to switch from proliferation to differentiation; how Wnt signals integrate with muscle specific regulatory factors in this process is poorly understood. We previously demonstrated that the Barx2 homeobox protein promotes differentiation in cooperation with the muscle regulatory factor (MRF) MyoD. Pax7, another important muscle homeobox factor represses differentiation. We now identify Barx2,MyoD,and Pax7 as novel components of the Wnt effector complex, providing a new molecular pathway for regulation of muscle progenitor differentiation. Canonical Wnt signaling induces Barx2 expression in muscle progenitors and perturbation of Barx2 leads to misregulation of Wnt target genes. Barx2 activates two endogenous Wnt target promoters as well as the Wnt reporter gene TOPflash, the latter synergistically with MyoD. Moreover, Barx2 interacts with the core Wnt effectors β-catenin and TCF, is recruited to TCF/LEF sites, and promotes recruitment of β-catenin. In contrast, Pax7 represses the Wnt reporter gene and antagonizes the activating effect of Barx2. Pax7 also binds β-catenin suggesting that Barx2 and Pax7 may compete for interaction with the core Wnt effector complex. Overall, the data show for the first time that Barx2, Pax7, and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts and that Barx2 and Wnt signaling participate in a regulatory loop. We propose that antagonism between Barx2 and Pax7 in regulation of Wnt signaling may help mediate the switch from myoblast proliferation to differentiation. RNA-Seq analyses was used to characterize gene expression in primary myoblasts from wild-type and Barx2 knockout mice.
Project description:Pax7 is a paired box transcription factor that is central to skeletal muscle satellite cell function Microarrays were used to examine gene expression after primary murine satellite cell-derived myoblasts were retrovirally infected with constructs encoding Pax7, PAX7-FOXO1A, dominant negative Pax7-ERD and retroviral control. Overall design: Primary murine satellite cell-derived myoblasts were expanded and then divided and each infected with a different construct. After a further 48 hours in culture, RNA was isolated.
Project description:GATA4 occupancy on the mouse genome of satellite cell-derived primary myoblasts. Proliferating myoblasts cultured in growth medium were immunoprecipitated with anti-GATA4 antibody or control IgG. Precipitated genomic DNAs were subjected to next generation sequencing. Paired-end 150 bp sequence reads of GATA4-ChIP and IgG-ChIP using mouse skeletal muscle myoblasts.
Project description:DUX4 is generally agreed to underlie pathology in Facioscapulohumeral muscular dystrophy Microarrays were used to examine gene expression after primary murine satellite cell-derived myoblasts were retrovirally infected with constructs encoding DUX4, DUX4c, constitutively active, dominant negative and truncated DUX4 versions. Overall design: Primary murine satellite cell-derived myoblasts were expanded and then divided into six sister cultures and each infected with a different construct. After a further 20 hours in culture, RNA was isolated.
Project description:Canonical Wnt/β-catenin signalling is an essential regulator of various cellular functions throughout development and adulthood. Aberrant Wnt/β-catenin signalling also contributes to various pathologies including cancer, necessitating an understanding of cell context dependent mechanisms regulating this pathway. Since protein-protein interactions underpin β-catenin function and localization, we sought to identify novel β-catenin interacting partners by affinity purification coupled with tandem mass spectrometry in vascular smooth muscle cells (VSMCs), where β-catenin is involved in both physiological and pathological control of cell proliferation. Here, we report novel components of the VSMC β-catenin interactome.