Investigating the Role of Meox2 in Myogenic and Tendon Differentiation

Correct development of the musculoskeletal system relies on intricate signaling pathways and gene regulation that facilitate cell differentiation.  Meox homeodomain transcription factor proteins are necessary for the development of the musculoskeletal system; mouse mutants for both Meox genes lack an axial skeleton and skeletal muscles of the trunk and limbs. Additionally, Meox2 mutants have a severe defect in limb tendon development. The aim of this study was three-fold, firstly to identify new regulators of tendon development and to analyze their expression in Meox2 mutants, secondly, to analyze the intrinsic regulation of myogenic differentiation by Meox2 transcription factor, and thirdly to establish an in vitro assay to analyze tendon fibroblast-myoblast interactions.  Firstly, literature based research identified genes that are expressed in tendon development in the chick embryo. Using a PCR based strategy; antisense RNA probes were generated, and used to analyze gene expression in mouse embryos. These experiments identified the Fibin2 gene as a novel marker for mouse tendon development, and a potential downstream effector of Meox2. Secondly, C2C12 myoblasts were transfected with plasmids encoding either native or dominantly active, or dominantly inactive variants of Meox2 protein. These experiments demonstrated that expression of any of the Meox variants strongly inhibited myogenic differentiation, as measured by the fusion index. This suggests that Meox proteins are essential for maintaining myoblasts in a progenitor state. Thirdly, tendon fibroblasts were isolated from young adult mice by collagenase treatment of the Achilles tendon using two published protocols. This was unsuccessful, while isolation of skin fibroblast was successful.  Further modifications are required to produce an effective protocol. The tendon fibroblasts we attempted to isolate are from Scleraxis–GFP transgenic mice, so all tendon cells are GFP positive. To label myoblasts for use in the tendon fibroblast-myoblast co-cultures, we investigated the potential use of a variety of fluorescently labeled cellular reporters, such as: Golgi-GFP, nuclear histoneH2B-Cherry, and membrane localized CAAX-Cherry. Plasmids encoding these reporters were transfected into C2C12 myoblasts and fluorescence was analyzed as these cells underwent in vitro differentiation. Initial experiments show strong expression of nuclear-Cherry, and weak expression of Golgi-GFP. Membrane localized CAAX-Cherry showed expression in multiple membrane compartments, not only the plasma membrane. As C2C12 cells differentiated the fluorescence of all reporters diminished. This was likely due to the fact that we used transiently transfected cells, and so we started selection of stably transfected cells to isolate clones, which show permanent, and strong gene expression to carry out future experiments.