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Fig. 2. Wnt signaling controls LR asymmetric gene expression. (A-P) Whole-mount in
situ hybridization analysis of Nodal expression (see text for
details). Headfold-stage wild-type (A,E), and Wnt3a-/-
(B,F) embryos express Nodal (purple) in the node. Two-color
whole-mount in situ hybridization showing Nodal (orange), and
Lefty1 and Lefty2 expression (purple) in wild-type (C,G) and
mutant (D,H) three-somite stage embryos. (G,H) High-power ventral views of the
nodes of the wild-type and mutant embryos depicted in C,D. Nodal
expression in wild-type four-somite (I,M) and similarly staged
Wnt3a-/- (J,N) embryos. Arrows in N indicate bilateral
Nodal expression in the mutant posterior LPM. Nodal was not
expressed in the wild-type LPM after the six- to seven-somite stage (K,O), but
was bilaterally expressed in the mutant LPM (L,P). (Q-S) Similar abnormal
expression patterns were observed for Lefty2 in the seven-somite
stage mutants (right embryos in Q and R, compare with the four-somite
wild-type embryos on the left), and for Pitx2 expression in
six-somite mutants (S). (T,U) Two-color whole-mount in situ hybridization
showing Nodal (purple) and Wnt3a (orange) expression in
four-somite wild-type (T) and AxinTg1/Tg1 (U) littermates.
(V,W) Lefty1/2 (purple) and Nodal (orange) expression in
two-somite wild-type (V) and AxinTg1/Tg1 (W) littermates.
Asterisks indicate ectopic bilateral expression of Nodal (U) and
Lefty1/2 (W) in anterior domains. (A-D,I-L,Q,T-W) lateral views;
(E-H,M,N,S) ventral posterior views; (O,P,R) anterior views; arrows indicate
the LPM; arrowheads indicate the anterior limit of LPM expression. ps,
primitive streak; n, node.
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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).
