The early and late G1 foci coincide with the Wnt reporter expression. (A) Analysis of Tcf/Lef:H2B-GFP reporter expression (nuclear GFP green) in fixed E12.5 and E13.5 whole-mount explants immunostained with an epithelial membrane marker EpCam to visualize the epithelial tissue (red). At E12.5, the Wnt reporter was expressed at the anterior part of the bud, and at E13.5 a new area of GFP-positive cells was seen at the tip of the bud. (B) 3D volume rendering showed the two separate areas of GFP positivity in the E13.5 tooth bud (early expression marked with an arrowhead and late expression with an asterisk). (C) The Tcf/Lef:H2B-GFP together with Fucci G1 showed colocalization in the early signaling center cells at E12.0, although GFP positivity was more widespread along the placode. At E13.0, the Tcf/Lef:H2B-GFP positive domain displayed a triangular shape similar to the G1 focus. At E13.5, a new G1 focus (the enamel knot) was apparent at the tip of the bud. (D) A schematic representation of the different cell populations in the forming bud. Bars: (A) 50 µm; (C) 40 µm.