Expression data from primary myoblasts with active Beta-catenin
ABSTRACT: 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.
INSTRUMENT(S): [MoGene-2_0-st] Affymetrix Mouse Gene 2.0 ST Array [mogene20st_Mm_ENTREZG_18.0.0]
Project description: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.
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: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:Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to Dystrophin-expressing myofibers upon transplantation, a subset of which maintain Pax7 expression in vivo and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that iMPCs can be established from muscle tissue following small molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple differentiated cell types. Overall design: 5 samples were analyzed in total: MEFs, MEFs+MyoD (day 14 of reprogramming), MEFs+MyoD+F/R (day 14 of reprogramming), iMPC clone and C2C12 myoblasts.
Project description:The mechanisms by which Pax7 promotes skeletal muscle stem (satellite) cell identity are not yet understood. We have taken advantage of pluripotent stem cells wherein the induced expression of Pax7 robustly initiates the muscle program and enables the generation of muscle precursors that repopulate the satellite cell compartment upon transplantation. Pax7 binding was excluded from H3K27 tri-methylated regions, suggesting that recruitment of this factor is circumscribed by chromatin state. Further, Pax7 binding provoked localized chromatin remodeling, including the acquisition of enhancer-associated histone marks and induction of chromatin accessibility. Conversely, removal of Pax7 led to rapid reversal of these features on a subset of enhancers. Another cohort of Pax7 binding sites exhibited a durably accessible and remodeled chromatin state after Pax7 removal, and persistent enhancer accessibility was associated with subsequent binding by muscle regulatory factors. Our studies provide new insights into Pax7 and the epigenetic landscape of skeletal muscle stem cells. Overall design: Examining the role of Pax7 in myogenesis and the chromatin context of Pax7-bound regions. (1) RNA-seq in the presence and absence of doxycyline (dox, inducing Pax7-expression) through the inducible iPax7-cell system (2) ChIP-seq of Pax7, H3K27Ac, H3K4me1, H3K4me3, and H3K27me3 in the presence and absence of dox in iPax7 myogenic progenitors (3) ATAC-seq of satellite cells, myogenic progenitors, myoblasts
Project description:Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to Dystrophin-expressing myofibers upon transplantation, a subset of which maintain Pax7 expression in vivo and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that iMPCs can be established from muscle tissue following small molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple differentiated cell types. Overall design: Examination of the transcriptome of induced myogenic progenitor cells in comparison to satellite cells
Project description:Abstract Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via interaction with Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analysed Taz in vivo and ex vivo in comparison to Yap. Taz was expressed in activated satellite cells. siRNA knockdown or constitutive expression of wildtype or constitutively active TAZ mutants showed that TAZ promoted proliferation, a function that was shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene symbol Wwtr1-/-) knockout mice, there were no overt effect on regeneration. However, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapflox/flox : Rosa26Lacz mice produced a marked regeneration deficit. To identify potential mechanisms, microarray analysis showed many common Taz/Yap targets, but Taz also regulates some genes independently of Yap, including myogenic genes such as Pax7, Myf5 and Myod1. Proteomic analysis of Yap/Taz revealed many common binding partners, but Taz also interacts with proteins distinct from Yap, that are mainly involved in myogenesis and aspects of cytoskeleton organization. Neither TAZ nor YAP bind members of the Wnt destruction complex but both extensively changed expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to promote myogenic differentiation.
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.