Figure 2. Msgn1 is directly activated by the Wnt/β-catenin pathway and Tbx6.

(a) VISTA “peaks and valleys” plot⁵⁸ illustrating a comparative analysis of the mouse and human Msgn1 loci (blue) including 1 kb of the upstream regulatory region (pink). The percent of conservation (y axis) between the two organisms is plotted against the coordinates of the mouse sequence (x axis). The pink regions are conserved non-coding regions, and the blue region is the conserved Msgn1 exon. The position and orientation of conserved consensus Tcf/Lef (red bars) and Tbox (green bar) binding sites identified in the promoter are displayed below the VISTA plot. Asterisks indicate nucleotides mutated for EMSA and transgenic reporter assays. (b) EMSA assay demonstrating the specific binding of Lef1 protein to Tcf/Lef BS probes but not to probes carrying mutated Tcf/Lef sites (asterisks). Lef1 binding to wildtype probes is competed by the addition of 125 times excess unlabeled probe. (c) Luciferase reporter assays for 1kb of the mouse Msgn1 promoter. Transfection of 293 cells with ΔN-β-catenin, or Tbx6-VP16 alone moderately activated the wild-type 1kb Msgn1 promoter, whereas cotransfection of both ΔN-β-catenin and Tbx6-VP16 resulted in a 7-fold activation. Coexpression of T-VP16 with ΔN-β-catenin did not result in a similar effect. Point mutations in all three Tcf/Lef BS abolished the activation of the luciferase reporter by Tbx6-VP16 and/or ΔN-β-catenin. (d) Representative transient transgenic embryos expressing the 1kb wildtype Msgn1 promoter-lacZ reporter (left) or a mutant version carrying mutations in all three Tcf/Lef BS (right). The fraction (bottom left corner) represents the number of embryos displaying the demonstrated pattern/total number of βgal positive transgenic founders. (e,f) 2 color WISH of Msgn1 (purple) and Tbx6 (orange) coexpression in E9 PSM, (e) and (f) are dorsal and lateral views, respectively. Tbx6 expression extends beyond the anterior border of Msgn1 expression. (g,h) Dorsal-posterior views of Msgn1 mRNA in E8.5 wildtype littermate controls (g) and Tbx6^−/− (h) embryos, oriented with posterior pointing down. Abbr.: BS, binding site; LOF, loss of function; GOF, gain of function; PS, primitive streak; PSM, posterior presomitic mesoderm. Scale bar = 100 μm.