Figure S1.
β-catenin induces apical localization of aPKC through its binding to N-cadherin. (A–J) HH23 chicken NTs 48 hpe with different molecular tools intended to independently manipulate the transcriptional (Transc) and structural (Struc) activities of β-catenin. The expected effect caused by each tool on the transcriptional and structural activities is indicated to the left of each row as follows: ↑, increased activity; ↓, decreased activity; ↑↓, no change. In addition, pCIG stands for the empty vector; β-catenin is a stable form of β-catenin (β-cateninS33Y), Sh-Scr and Sh-CTNNB1 are scrambled and anti-β-catenin inhibitory shRNAs, respectively. Tcf3·Vp16 is a constitutive activator of TCF-dependent transcription, and N-cadherinΔβcat is the N-cadherin mutant lacking the β-catenin–binding domain. The figure shows representative images of the transfected NTs used for the quantification in Fig. 2, and the channels are shown separately in grayscale for clarity. In panels A, C, E, G, and I, the slices were stained for F-actin (phalloidin, red), β-catenin (green), and GFP (blue, indicating transfection). Panels B, D, F, H, and J were stained for N-cadherin (red), aPKC (green), and GFP (blue, indicating transfection). The arrows in panels E, E’, and E’’ indicate Sh-CTNNB1–transfected cells with a round morphology, some of them protruding into the ventricle. Knockdown of β-catenin by Sh-CTNNB1 is evident in panel E’’. In panel F, the arrowheads and arrow point to round and protruding cells, respectively. In panels G and H, the arrows pinpoint small invaginations caused by Tcf3·Vp16, while the arrows in panel H’’ indicate the accumulation of cytoplasmic aPKC in the transfected areas. Arrows in panel I and I’ point to cells protruding into the ventricle, and the arrows in panel J’’ denote the accumulation of cytoplasmic aPKC in cells expressing a combination of Tcf3·Vp16 and Sh-CTNNB1. Note that in this case, the knockdown of β-catenin caused a loss of apico-basal polarity rather than the typical small invaginations normally induced by Tcf3·Vp16. (K and L) HH12 chicken NTs transfected with wild-type N-cadherin (K) or N-cadherinΔβcat (L) stained with antibodies against N-cadherin (red) and aPKC (green) 48 h later. GFP is shown in blue, indicating transfection. (M) Knockdown efficiency of two different inhibitory shRNAs targeting CTNNB1 studied by quantitative RT-PCR (RT-qPCR) in chicken embryonic fibroblasts (CEFs). Each experimental condition was compared with its control condition using an unpaired t test (n = 3). (N) Wnt pathway activity (TOPFlash) studied by a luciferase reporter assay in HH18 chicken NTs 24 hpe with control, Sh-CTNNB1, β-catenin, Sh-CTNNB1 plus β-catenin, Tcf3·Vp16, or Tcf3·Vp16 plus Sh-CTNNB1. Bar graphs show the mean ± SD. Each experimental condition was compared with every other experimental condition using a one-way ANOVA with Tukey’s multiple comparisons test (n = 10). *, P < 0.05; **, P < 0.01; ***, P < 0.001. cad, cadherin.