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An integrated strategy for analyzing the unique developmental program of different myoblast subtypes

ABSTRACT: An important but largely unmet challenge in understanding the mechanisms that govern formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from twelve genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets—based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes—provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system. Keywords: repeat

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE3854 | GEO | 2006/01/01

SECONDARY ACCESSION(S): PRJNA94081

REPOSITORIES: GEO

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Project description:An important but largely unmet challenge in understanding the mechanisms that govern formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from twelve genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasetsâ??based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes ?provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system.

Project description:Wnt/M-NM-2-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and M-NM-2-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 M-NM-2-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 M-NM-2-catenin/Tcf complex formation, reduced basal M-NM-2-catenin in cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased membrane-bound M-NM-2-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated M-NM-2-catenin (Tyr654) during myogenic differentiation. These results suggest that various Wnt ligands control subcellular M-NM-2-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via M-NM-2-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.

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An integrated strategy for analyzing the unique developmental program of different myoblast subtypes

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