(a) Scheme of the different DNA constructs used in the figure, all constructs except the reporters (TopFlash and TopRed), were cloned into pCIG. Modulators of Tcf-dependent transcription contained a Tcf3-HMG box alone (Tcf3-HMGB) or that was fused to the VP16 activator (VP16-Tcf3) or the Engrailed repressor (EnR-Tcf3). TopFlash and TopRed reporter constructs contained a 5 × Tcf-binding motive controlling luciferase and RFP, respectively. The transcriptional strength of β-cateninS33Y and Vp16·Tcf3 was compared with TopFlash in HH-12 chicken embryos. The performance of TopRed was assessed in human embryonic kidney-293 with VP16·Tcf3 and EnR·Tcf3. (b–h) The different construct combinations were analysed 48 h after electroporation into HH-12 chicken neural tubes. Transverse sections were stained with antibodies against GFP (green, transfection), RFP (red, Tcf-dependent activity) and N-cadherin (blue). The green channel has been omitted in the picture at the right of each panel for clarity. Arrows indicate the invaginations. The absence of α-catenin binding by the mutant used in panel e (β-cateninS33YΔαCatBs) is shown in Supplementary Fig. 6. Scale bar, 50 μm.