Fig. 3. Examination of cilia in the Wnt3a^-/- mutants. (A,B) High-power SEM micrographs of cilia in wild-type (A) and Wnt3a^-/- (B) nodes of head-fold stage embryos (E7.75-8). (C,D) Projection images of node cilia visualized using anti-acetylated tubulin antibodies. (E) Two-color whole-mount in situ hybridization analysis of Lefty1 (orange) and Lrd (purple) expression in three-somite wild-type (left) and Wnt3a^-/- (right) embryos. (F) Wnt3a (orange) and Nodal (purple) expression in three iv/iv embryos. (G-Q) Confocal microscopy images of PC1 (G,K), PC2 (H,L), acetylated tubulin (I,M) and the merged images (J,N,O,P,Q) in E7.75 wild-type (G-J,O) and Wnt3a^-/- (K-N,P,Q) nodes. The abundance of white spots in global views of the node indicated co-expression of all three markers in individual wild-type cilium (arrows, G-J). By contrast, Wnt3a^-/- cilia predominantly appeared as purple spots (N), indicating co-expression of only PC2 and tubulin. Views were selected to present sufficient numbers of labeled cilia and therefore depict slightly different regions of the wild-type and mutant node; however, images are representative of the entire node. Profiles of labeled cilia illustrate the co-expression of PC1 and PC2 in distinct spatial domains in wild-type cilia (arrows, O). Rare mutant cilia co-expressed PC1 and PC2 but these domains were unusually small and not easily detected (arrow, P). Cilia co-expressing PC2 and tubulin were easily detected in the mutant node (Q).