Electroporations were performed in HH-12 chicken embryos. Scale bars represent 10 μm in c,d,f,g,i and 50 μm in b–h. (a) Neural tubes were electroporated with control (pCIG) or β-cateninS33Y and AC proteins assessed by WB. Protein levels were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the difference between control and β-cateninS33Y was calculated. Fold change=mean of three experiments ±s.d. (b) Neural tubes were electroporated with β-cateninS33Y·FLAG and stained with antibodies against GFP (green, transfection), FLAG (red, β-cateninS33Y distribution) and N-cadherin (blue). (c–e) Representative high-magnification images and pixel intensity quantification (see Methods for statistics) of neural tubes transfected with β-cateninS33Y and stained against GFP (greys, transfection), ZO-1 (red in c) or aPKC (red in d) and N-cadherin (green). (f) Similar sections to those in c were stained against GFP (blue, transfection), aPKC (green) and P-aPKC (red). The separate channels are shown in grey for comparison. (g) Neural tubes were electroporated with β-cateninS33Y and stained against GFP (green, transfection), N-cadherin (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue, labels DNA). The three panels show β-cateninS33Y+ cells at three different stages of the cell cycle. In the early mitosis panel, two cells with condensed chromosomes are indicated (arrowhead=non-transfected, arrow=β-cateninS33Y+). An image omitting the GFP channel is shown to the right of each panel for clarity. (h) Neural tubes were electroporated with Eng·Tcf3 plus a dominant negative Tcf4 (DN Tcf4, a scheme of the constructs is shown to the right of the pictures), sections were stained against GFP (blue, transfection), aPKC (red) and N-caderin (green). Arrowheads point to discontinuities in the ACs coincident with transfected cells. Two enlarged images showing areas of transfected cells with AC deficiencies (arrowheads) are shown at the right of the pannel. (i) Very high-magnification image of a section as in h, ZO-1 is mislocated in cells with no N-cadherin at the AJs (arrowhead). Sections stained with GFP (blue, transfection), ZO-1 (red) and N-cadherin (green).