Tissue formation during development involves the determination, controlled proliferation and specific differentiation of cells in the embryo. Misregulation in any phase of this process can lead to failure in the development of the embryo, severe disease or uncontrolled cellular gorwth. Thus the study of gene regulation during development provides insight into areas important in human disease. Embryonic muscle formation in vertebrates and Drosophila (the fruit fly) provides an excellent model system in which to study the origin of one of the major tissues in higher organisms. The determination, proliferation, and differentiation of muscle cells during development in both vertebrates and invertebrates depend upon the function of the MyoD family of basic helix-loop-helix proteins (MRFs). Determination of the first muscle precursor cells involves the activation of the MRFs in early mesoderm while gene expression characteristic of differentiated muscle remains repressed. Terminal differentiation is marked by the withdrawal of the myoblast from the cell cycle just prior to the activation of the muscle-specific genes and both processes involve the MRFs. Cell cycle control during terminal differentiation is thought to involve MRF regulation of the phosphorylation status of the retinoblastoma protein, Rb. (Project 1) We have recently shown that MyoD binds directly to the G1 cyclin-dependent kinase cdk4 to inhibit cell growth and the phosphorylation of Rb while the cdk4-MyoD interaction blocks the trans activation functions of MyoD by disrupting DNA-binding of the MyoD/E-protein heterodimer. Therefore, high levels of nuclear cdk4 can block MyoD function in growing myoblasts while the loss of nuclear cdk4 in the absence of growth factors allows MyoD to function. The 15 amino acid domain on MyoD responsible for interaction with cdk4 is sufficient for these effects. Expression of this domain as a fusion protein with GST or GFP inhibits cell growth and induces myoblast differentiation under growth conditions. We have a patent application on the inhibitory activity of the 15 amino acid domain of MyoD on cdk4 kinase activity. We have recently made the 15 amino acid cdk4 binding domain of MyoD with alanine residues in each position in order to map the residues involved in cdk4 binding. This is being checked with cdk4 produce in the baculovirus system. The binding affinities are to be measured on the BiaCore. These results are also being used to look at cdk6 and cdk2 interactions with the same domain of MyoD. In Drosophila we have also shown that MyoD (nautilus) expression defines a subset of mesodermal cells that are required to set up the muscle pattern in each hemisegment of the embryo. Toxin ablation of nautilus positive cells, induced antisense expression to nautilus RNA, or injection of double stranded nautilus RNA into the embryo (RNA interference or RNA-i) all eliminate muscle formation in the embryo and define nautilus as an essential gene for myogenesis in the fly. This study demonstrated the general utility of RNA-i ablation of gene function in Drosophila in the absence of a genetic mutation in the gene of interest. We have also established the method in cultured Drosophila Schneider cells. RNA-i does not appear to work in mammalian cells , as yet, even if we block PKR activity and the induction of the interferon pathway by using PKR -/- cells and the PKR inhibitor, p58. ES cells are also very succeptible to killing by dsRNA so developmental stage does not appear to be a key factor. (Project 2) Determination of the myoblast in the mesoderm involves the activation of MyoD and MyoD responsive downstream target genes. We have been studying the activation of the single MyoD gene, nautilus, in Drosophila and have determined that the nautilus promoter is activated predominately by DMEF2, a Drosophila SRF homolog, and twist, a major determinant of the mesoderm. We can induce Schneider cells to activate a partial myogenic program by expressing daughterless in these cells. This myogenic conversion is potentiated by the coexpression of DMEF2 and nautilus. The cells exit the cell cycle, become multinucleated and express myosin. Myogenic conversion of Schneider cells is dependent upon the endogenous expression of nautilus and DMEF2, two mesodermal markers that establish for the first time that Schneider cells are of mesodermal origin. Endogenous gene function for nau, DMEF2 and other genes was inhibited using RNA interference. Daughterless conversion is driven by ectopic daughterless expression due to the fact that Schneider cells express 100-1000 fold less daughterless than nautilus. Raising the levels of daughterless protein allow sufficient levels of the nau/da heterodimer to form to activate the myogenic program. This work helped to define conditions for the application of RNAi to cultured Drosophila cells and defined a partial myogenic system allowing one to analyze nautilus-responsive genes by differetial display. This RNA interference assay is being used to explore the role of other factors in the myogenic process as well. We hope to analyze genes that are differentially expressed between the non-myogenic and myogenic state to identify early target genes for myogenic conversion. In addition, these results support our conclusions regarding the essential nature of the MyoD homolog, nautilus, in Drosophila myogenesis.
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