The Transcriptional Regulation of Premyogenic Mesoderm Factors during Skeletal Myogenesis

Abstract

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal myocytes from the undifferentiated state. The present study aims to understand and compare the transcriptional regulation of skeletal muscle progenitor and MRFs through beta-catenin- and MyoD-induced P19 EC cell differentiation. Furthermore, the study aims to compare the expression pattern of transcription factors that mark either the epaxial or hypaxial domain of the differentiated somite of the developing embryo in DMSO-, beta-catenin- and MyoD induced P19 EC cell differentiation. First, stable expression of an active beta-catenin in P19 cells was sufficient to induce the expression of the premyogenic mesoderm factors such as Pax3/7, Meox1, Gli2, Foxc2, in addition to the somitic marker Foxc2. Knock-down of beta-catenin, by shRNA, resulted in a decreased expression of these factors, and a downregulation of the MRFs, indicating that beta-catenin is essential for myogenesis. DMSO-induced differentiation led to the upregulation of Sim1 and Lbx1, whereas beta-catenin-induced differentiation did not show any significant changes to either. Second, gain- and loss-of-function experiments demonstrated that MyoD directed P19 cells into the skeletal muscle lineage by inducing the expression of Meox1, Pax7, Six1, and Eya2, on day 4 and eventually Pax3 leading to the subsequent upregulation of the MRFs. Chromatin immunoprecipitation experiments showed that MyoD bound directly to the regulatory regions of Pax3/7, Eya2, Six1, Meox1 and myogenin. Furthermore, MyoD-induced differentiation upregulated the expression of Lbx1 and C-met and had relatively unchanged levels of En1 and Sim1 expression. These results demonstrate that MyoD re-directs P19 cells into the skeletal muscle lineage by directly binding to the regulatory regions of several premyogenic mesoderm genes, leading to the formation of skeletal muscle that express the transcription factors C-met and Lbx1, both of which are shown to be essential in limb, diaphragm and tongue muscle formation in the embryo. These studies have provided an understanding of the transcriptional regulation of premyogenic mesoderm factors in both beta-catenin- and Myod-induced differentiation, with further insight into the type of skeletal muscle that may be forming in P19 EC cells.