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β-Catenin Is Essential for Differentiation of Primary Myoblasts via Cooperation With MyoD and α-Catenin

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β-Catenin Is Essential for Differentiation of Primary Myoblasts via Cooperation With MyoD and α-Catenin

Shuang Cui et al. Development.

Abstract

Canonical Wnts promote myoblast differentiation; however, the role of β-catenin in adult myogenesis has been contentious, and its mechanism(s) unclear. Using CRISPR-generated β-catenin-null primary adult mouse myoblasts, we found that β-catenin was essential for morphological differentiation and timely deployment of the myogenic gene program. Alignment, elongation and fusion were grossly impaired in null cells, and myogenic gene expression was not coordinated with cytoskeletal and membrane remodeling events. Rescue studies and genome-wide analyses extended previous findings that a β-catenin-TCF/LEF interaction is not required for differentiation, and that β-catenin enhances MyoD binding to myogenic loci. We mapped cellular pathways controlled by β-catenin and defined novel targets in myoblasts, including the fusogenic genes myomaker and myomixer. We also showed that interaction of β-catenin with α-catenin was important for efficient differentiation. Overall the study suggests dual roles for β-catenin: a TCF/LEF-independent nuclear function that coordinates an extensive network of myogenic genes in cooperation with MyoD; and an α-catenin-dependent membrane function that helps control cell-cell interactions. β-Catenin-TCF/LEF complexes may function primarily in feedback regulation to control levels of β-catenin and thus prevent precocious/excessive myoblast fusion.

Keywords: Cell signaling; Differentiation; Epigenetics; Homeobox genes; Muscle stem cells; Myogenesis; Skeletal muscle; Transcription factors.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Characterization of β-catenin-null primary myoblasts. (A) Strategy for Cas9/gRNA-mediated mutation of β-catenin. The blue nucleotides represent the gRNA target sequence; the highlighted nucleotides represent the protospacer adjacent motif (PAM). (B) β-Catenin mRNA expression was measured by RT-PCR in multiple independent CRISPR-generated myoblast lines and compared with that in wild-type myoblasts transfected with the empty vector (LentiCRISPRv2). Data are normalized to the housekeeping gene RPS26 and show the average of two experiments performed in duplicate. Statistical analysis: one-way ANOVA (*P<0.05). (C) Western blot analysis using β-catenin antibody shows that β-catenin-null primary myoblasts are completely devoid of β-catenin protein expression. β-Actin was used as a loading control. (D) β-Catenin-null myoblasts show no induction of Axin2 mRNA after treatment with Wnt3a for 24 h. Statistical analysis: t-test (****P<0.05). (E) Immunofluorescence labeling of β-catenin (red) in cultured wild-type and β-catenin-null primary myoblasts; nuclei are stained with DAPI (blue). Scale bars: 25 µm.
Fig. 2.
Fig. 2.
Differentiation capacity of wild-type and β-catenin-null primary myoblasts. (A) Phenotype of wild-type and β-catenin-null primary myoblasts cultured in growth media (GM) showing lack of spontaneous differentiation. (B) Phenotype of wild-type and β-catenin-null primary myoblasts after 5 days of culture in Wnt3a-containing media showing greatly impaired differentiation. Scale bars: 100 µm.
Fig. 3.
Fig. 3.
Immunostaining analysis of myogenic markers in wild-type and β-catenin-null primary myoblasts after Wnt3a treatment. (A,C) Immunofluorescence staining for myogenin (A) or MyHC (C), both green. Nuclei were stained with DAPI (blue). Images were captured at 0, 24, 48, 72 and 120 h post-treatment. (B,D) Quantification of myogenin- (B) or MyHC- (D) positive cells at each time point. Data are mean±s.e.m. Statistical analysis: one-way ANOVA (****P<0.0001).
Fig. 4.
Fig. 4.
Transcriptomic analysis of wild-type and β-catenin-null cells after Wnt3a treatment identifies the Wnt-induced gene set. (A,B) RNA-seq was performed on wild-type (A) and β-catenin-null (B) myoblasts treated with Wnt3a or control (L-cell) medium for 24 h. Genes upregulated by Wnt3a treatment (Wnt3a/L cell≥twofold, red nodes) were analyzed using Toppcluster to define associated pathways (green nodes). Network analysis of global gene expression changes in wild type shows many genes upregulated by Wnt3a in wild type but not in β-catenin-null myoblasts. (C,D) Gene Ontology (GO) terms significantly enriched in the differentially expressed gene set (Wnt3a/L cell≥twofold) identified in wild-type (C) and β-catenin-null (D) myoblasts. (E) Wnt3a induces expression of myogenic genes myomaker (Tmem8c), Fst and myogenin in a β-catenin-dependent manner. Quantitative RT-PCR was performed on wild-type and β-catenin-null myoblasts treated with Wnt3a-containing or control (L-cell) medium for 24 h. Data are normalized to the housekeeping gene RPS26 and are the average of three experiments performed in duplicate. Data are mean±s.e.m. Statistical analysis: t-test (*P<0.05, **P<0.01).
Fig. 5.
Fig. 5.
Epigenomic analyses indicate that β-catenin promotes MyoD association with chromatin. (A) H3Kac and H3K4me3 ChIP-seq was performed using chromatin from wild-type myoblasts treated with control or Wnt3a-containing media for 24 h. Homer analysis was used to identify the top 10 motifs that are enriched in promoter regions that show increased levels of H3Kac and H3K4me3 (Wnt/L cell≥threefold) after Wnt3a induction (represented as target sequences with motif/total sequences with motif). (B) MyoD ChIP-seq analyses of wild-type and β-catenin-null primary myoblasts treated with Wnt3a medium for 24 h. Gene ontology (GO) analysis was performed on the set of genes showing increased MyoD binding after Wnt3a treatment (Wnt/L cell≥twofold) in wild-type (left) and β-catenin CRISPR-null (right) myoblasts. Wild-type myoblasts show significant enrichment of muscle-associated GO categories; β-catenin-null myoblasts do not show enrichment in any muscle-specific categories. (C) The top 15 motifs (Homer analysis) seen in promoter regions showing increased MyoD binding (Wnt/L cell≥twofold) after Wnt3a treatment of wild-type myoblasts (target sequences with motif/total sequences with motifs). Statistical analysis: multiple t-tests with P<0.05 considered significant. (D,E) Wnt3a increases binding of β-catenin and MyoD to the proximal promoters of the myomaker and myogenin genes, as shown by ChIP-qPCR analysis. Chromatin from wild-type primary myoblasts treated with control or Wnt3a-containing medium for 48 h was used for ChIP with β-catenin (D) or MyoD (E) antibodies. qPCR was used to assess enrichment of the myomaker promoter in both β-catenin- and MyoD-ChIP samples; the myogenin promoter was used as a control for MyoD-ChIP and the Axin2 enhancer was used as a control for β-catenin-ChIP. Data were first normalized to a control non-target locus (rDNA1) and then to the mock ChIP with preimmune IgG, set to a value of 1. n=3. Data are mean±s.e.m. *P<0.05, **P<0.001.
Fig. 6.
Fig. 6.
Comparison of the ability of wild-type and mutant forms of β-catenin to rescue differentiation of β-catenin-null myoblasts. (A) β-Catenin-null myoblasts were co-transfected with GFP and one of the following expression plasmids: empty pcDNA3, an α-catenin-binding site (BS) mutant form of β-catenin (which cannot interact with α-catenin), a TCF-binding site (BS) mutant form of β-catenin (which cannot interact with TCF/LEF) and wild-type β-catenin (constitutively active/stable). Scale bars: 50 µm. (B,C) Quantitation of differentiation was achieved by immunostaining with MyHC (B) and counting the percentage of nuclei (blue, DAPI) in MyHC-positive myofibers (C). Data are mean±s.e.m. Statistical analysis: one-way ANOVA (*P<0.05, ****P<0.0001).

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