Fig. 8. T-box mediated anterior repression of vab-7. (A) Upper line, schematic of vab-7 5' regulatory region of vab-7::GFP construct showing putative T-box binding sites A and B. Lower line, sequence of mutated sites A and B, following site directed mutagenesis. These mutated sites would not be expected to bind T-box proteins (Sinha et al., 2000). (B,D,F) Expression of vab-7::GFP in transgenic animals carrying this mutated construct. The normal posterior pattern of vab-7::GFP expression can be seen, together with ectopic expression at the anterior. (B) Ectopic anterior expression can be seen at around the 100-cell stage (arrows). The inset panel in (C) shows WT vab-7::GFP expression in four posterior Cxxp cells at the same stage, for comparison. Note there is no anterior expression in this case. (D) Ectopic anterior expression around the 400-cell stage (arrows) together with the usual posterior muscle expression of vab-7::GFP seen at this stage. (F) Ectopic expression at the anterior at the 1.5-fold stage (arrows) together with the usual posterior muscle and hypodermal VAB-7::GFP observable at this stage. (C,E,G) expression of WT, non-mutated vab-7::GFP in tbx-30(RNAi) embryos. Ectopic expression of vab-7 is observable in the same anterior cells at comparable stages to the T-box binding site-mutated vab-7 reporter, suggesting that TBX-30 normally represses vab-7 expression at the anterior of embryos by binding to these sites. (C) Ectopic expression of vab-7 around the 100-cell stage in the same cells as in (B) (arrows). The inset panel shows WT vab-7::GFP expression at the same stage, for comparison. (E) Ectopic anterior expression around the 400-cell stage (arrows), comparable to that seen in (D). (G) Ectopic anterior expression at the 1.5-fold stage, comparable to that seen in (F). The usual posterior vab-7 expression pattern is seen in all cases (slightly out of focus in (G)) Posterior is to the right, dorsal is up in panels (B-G). Scale bar, 10 µm.