Fig. 3. Dominant-negative Brachyury (dnBrachyury; T-box domain) blocks BMP2-mediated chondrogenic development in C3H10T1/2 MSCs in vitro and ectopically in vivo. (A) Brachyury's T-box domain interferes with the transcriptional activity of full-length Brachyury. For the assessment of Brachyury's transcriptional activity, the Brachyury Binding Element (BBE) (see Materials and Methods) was placed twice in front of an HSV TK minimal promoter fused to a choloramphenicol-acetyltransferase reporter gene. These constructs were transfected into HEK293T cells with a full-length Brachyury expression construct and increasing amounts of an expression construct encoding the T-box domain (dnBrachyury). The total amount of Brachyury expression constructs was kept constant or filled up with empty vector (control). The ratios of full-length BrachyurydnBrachyury were increasing from 1:1 to 1:2 and 1:3, as indicated. Values represent -fold activation in relation to an empty vector and are the average of at least three experiments done in triplicate. (B) Expression of dnBrachyury (T-box domain) in C3H10T1/2-BMP2 during cultivation (day 0; cellular confluence) The T-box domain (aa 1-229) was subcloned and HA-tagged in expression vector pMT7T3 and constitutively expressed in C3H10T1/2-BMP2 cells. Western immunoblotting was with HA-antibody SC-805 (Santa Cruz). The recombinantly expressed T-box domain (dnBrachyury) is indicated (triangle). The marker was carbonic anhydrase (29 kDa). (C) RT-PCR experiments with osteo/chondrogenic marker genes show that T-box domain (dnBrachyury) expression in C3H10T1/2-BMP2 cells interferes with the BMP2-dependent FGFR2 but not FGFR3 expression. (D) dnBrachyury (T-box) interferes with BMP2-mediated FGFR2 expression, as analyzed by western immunoblotting with antiFGFR3 and antiFGFR2 antibodies, as described Fig. 1. (E) The forced expression of the dominant-negative acting T-box domain in C3H10T1/2-BMP2 cells interferes with BMP-2 mediated osteo/chondrogenic development. dnBrachyury interferes with expression of alkaline phosphatase-positive and with Alcian Blue-positive matrix synthesis of chondrocyte-like cells at days 6 and 11 post-confluence, respectively. dnBrachyury also interferes with BMP2-dependent osteo/chondrogenic development at murine ectopic sites after intramuscular transplantation (20 days after implantation; HE-staining) resulting in connective tissue formation at intramuscular sites, only.

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