Fig. 1. T-box binding sites are essential components of the Ciona Mesp regulatory elements. (A) Diagram depicting the two lineages derived from the B7.5 cells (TVC, trunk ventral cells). (B) Transgenic, gastrulating embryo expressing the 110-bp Ci-Mesp-lacZ reporter gene, hybridized with a probe against lacZ mRNA. (C) Transgenic tailbud embryo expressing the 110-bp Ci-Mesp-lacZ reporter gene, stained with X-gal. (D) Transgenic tailbud embryo expressing the 107-bp Ci-Mesp-lacZ reporter gene, stained with X-gal. (E) Upstream sequences for Ci-Mesp and Cs-Mesp; numbers indicate the distance from the putative transcription start site. Black bars indicate minimal 110- and 105-bp enhancers. Red bars represent the distal deletions that rendered the minimal enhancers inactive. Putative T-box binding motifs are boxed and lettered; matches to the Ciona Tbx6-binding site are highlighted in green lettering. Gray boxed areas indicate the only stretches of conserved sequence between the two enhancers. (F) Summary of expression obtained with Ci-Mesp-lacZ fusion genes. Putative T-box binding motifs are indicated by lettered boxes. The motif sequences below show matches to the Tbx6 consensus-binding site (Yagi et al., 2005) in green; mutated nucleotides for disruption of the binding motif are shown in red, whereas those for enhancement of the motif are in blue. +++, strong, consistent staining of B7.5 lineages; +, weak, inconsistent staining; 0, no staining. All results are representative of at least two trials and were unambiguous for the hundreds of embryos observed in each trial.