Fig 7. Tbx2 represses downstream targets of VegT and Brachyury.

(A) Tbx2 represses expression of derriere, xwnt11, and bix4 (Zhang and King, 1996; Casey et al., 1999). RT-PCR analysis of animal caps dissected at late blastula stages and cultured until mid-gastrula stages, as indicated. Activin (0.5ng/ml) was added to stage 8 animal caps, as indicated. (B) tbx2 represses expression of a Bix4 reporter construct. Embryos at the 2-cell stage were injected with bix4-LUC (20pg) and pRLTK (7pg), in the absence and presence of tbx2 RNA (250 pg). Whole embryo lysates from gastrula stage embryos were assayed in triplicate for Firefly and Renilla luciferase activity. (C) Mutation of three T-box binding sites renders bix4 insensitive to repression by Tbx2. Embryos were injected at the 2-cell stage with pRLTK (7pg) and bix4-LUC (20pg), Td-LUC (20pg), or Tdmp-LUC (20pg) in the absence and presence of tbx2 RNA. Tbx2 blocks the expression of Td-LUC less efficiently than it does bix4-LUC, while mutation of the three T-box sites renders Tdmp-LUC insensitive to repression by Tbx2. Samples of five whole embryo lysates from gastrula stage embryos were assayed in triplicate for Firefly and Renilla luciferase activity. Error bars indicate standard error.