Fig. 2. The T-box transcription factor Brachyury mediates chondrogenic differentiation in MSCs in vitro and ectopically in vivo. (A) Schematic representation of Brachyury according to Kispert et al. (Kispert et al., 1995). Western immunoblotting of recombinant HA-tagged Brachyury (aa 1-436) in cellular extracts of C3H10T1/2 (C3H10T1/2-Brachyury) with HA-antibody SC-805 (Santa Cruz) Brachyury is constitutively expressed under the control of the murine PGK-promoter. Expression of Brachyury is indicated (triangle). The molecular mass marker (M) shown is ovalbumin (43 kDa). NLS, nuclear localization signal. (B) Histological characterization of C3H10T1/2-Brachyury cells in culture. Top: at day 4 post-confluency cells develop alkaline-phosphatase-positive osteoblast-like cells. Below right: Alcian Blue histology of C3H10T1/2 cells stably expressing Brachyury show secreted proteoglycans and efficient differentiation into the chondrogenic lineage. Below left: Collagen-immunohistochemistry of C3H10T1/2-Brachyury cells in culture 7 days post-confluency was performed as described in Materials and Methods. Monoclonal anti-collagen 2 antibodies and anti-collagen X antibodies were used for collagen histology. The red precipitate of AEC (aminoethylcarbazole) indicates a positive signal. Cells secrete chondrocyte-specific collagen 2 but not collagen X, which is specific for hypertrophic chondrocytes. (C) RT-PCR analysis of the expression at various time intervals of chondrogenic and osteogenic marker genes in C3H10T1/2 cells recombinantly expressing Brachyury. (D) The forced expression of the T-box factor Brachyury in C3H10T1/2 cells leads to differentiation into chondrocytes and cartilage development at murine ectopic sites after intramuscular transplantation. Transplantations were performed as outlined in Materials and Methods. C3H10T1/2-Brachyury cells differentiate into chondrocytes after ectopic implantation at murine intramuscular sites (10 days and 20 days after implantation; HE, Alcian Blue and Safranin O staining). C3H10T1/2-Brachyury cells develop chondrocytes (20 days after implantation). Day 0, 1st day of cellular confluency.