Cfl1^C5 enhances the convergent extension defect in the Vangl2^Lp notochord. (A-D) Projections of flat-mounted E8.0 embryos stained for brachyury (T) (magenta). (A′-D′) Higher magnifications (4×) of midline cells from boxed regions of A-D highlight the arrangement of cells in each genotype. Wild type (A) and Cfl1^C5 (B) have a similar arrangement of T⁺ cells in the midline, whereas the Vangl2^Lp midline (C) is wider and shorter. The Vangl2^Lp Cfl1^C5 mutant midline (D) is wider and shorter than Vangl2^Lp. (E,F) The LWR of the notochordal plate measured by the distribution of cells (E) or in μm (F). Scale bars: 100 μm in A-D; 25 μm in A′-D′. Data are mean±s.d.

Defects in left-right asymmetry in Vangl2^Lp Cfl1^C5 double mutant embryos. (A-C) A fraction of Vangl2^Lp Cfl1^C5 double mutants examined at E9.5 showed reversed heart looping (arrow), a phenotype not seen in either single mutant. (D-I) This phenotype is due to an early defect in left-right asymmetry, as some double mutants express Nodal (purple) bilaterally in the lateral plate mesoderm and show increased Nodal expression on the right side of the node. Arrows indicate the side of the node with stronger Nodal expression. Scale bars: 100 μm.

Nodal cilia are not posteriorly positioned in Vangl2^Lp Cfl1^C5 double mutants. (A,C,E) Scanning electron microscope images of the node show that cilia are polarized to the posterior of node cells in wild type, Cfl1^C5 and Vangl2^Lp single mutants. (B,D,F) Basal bodies, visualized by the centrosomal protein pericentrin (green), show a similar posterior position in the cells of wild type, Cfl1^C5 and Vangl2^Lp single mutants. F-actin at the cell borders is highlighted with phalloidin (red). (G) In Vangl2^Lp Cfl1^C5 double mutants, many cilia point anteriorly (arrows).(H) Many basal bodies are mislocalized to the anterior half of the cell in Vangl2^Lp Cfl1^C5 double mutants (arrows). Anterior is towards the top in all images. (I) The relative localization of basal bodies in individual central node cells was quantified from immunofluorescence images from at least three embryos per genotype. The graph shows that basal bodies in double mutants are not positioned on the posterior of cells; in addition, there is a greater variance in the placement of cilia compared with wild type or either single mutant. Data are mean±s.d. Scale bars: 2 μm.

Celsr1, a core component of the PCP pathway, is not planar polarized in Vangl2^Lp Cfl1^C5 double mutants. (A-B) Celsr1 (green) is enriched on the anterior/posterior faces of node cells in wild-type embryos. Cell boundaries are highlighted by phalloidin (red), anterior towards the top.(C-F) The anterior/posterior enrichment of Celsr1 is retained in Cfl1^C5 mutants (C-D) and Vangl2^Lp mutants (E-F). (G-H) By contrast, Celsr1 is not enriched at the apical membrane in Vangl2^Lp Cfl1^C5 double mutants (G-H). (B,D,F,H) The mean intensity of Celsr1 staining normalized to phalloidin intensity along different edges of the cell. Celsr1 intensity was measured along individual cell borders and binned based on the angle with respect to the mediolateral axis of the embryo, such that 0-29° (horizontal) lines correspond to anterior/posterior faces of the cell, and 60-90° (vertical) lines correspond to mediolateral faces of the cell. Scale bar: 10 μm. Data are mean±s.d. ^*P<0.001.

Decreased cofilin activity is sufficient to disrupt apical trafficking of the PCP proteins Celsr1 and Vangl2. (A-A″) The PCP proteins Celsr1 and Vangl2 colocalize to the anterior/posterior faces of node cells in wild-type embryos. (B-B″) Celsr1 and Vangl2 colocalize in cytoplasmic puncta in the compound Cfl1^C5 Dstn^corn1/+ mutant. (C-H) Effects of pharmacological inhibitors on the planar polarization of PCP proteins in cultured embryos. (C-C″) Late headfold (LHF) stage embryos, shown before culture (C), show normal Celsr1 localization after 14-18 hours of culture in 10 nM jasplakinolide (C″). (D-D″) When cultured in the presence of jasplakinolide under the same conditions, earlier embryos (late-bud stage, LB) phenocopy Vangl2^Lp Cfl1^C5 and Cfl1^C5 Dstn^corn1/+, as Celsr1 fails to become membrane localized (D″). (E,E′) DMSO control for the late-bud stage culture. (G,G′) Blocking Wnt ligand secretion does not prevent recruitment of Celsr1 to the membrane, as embryos cultured with 5 μM IWP2 (an inhibitor of Wnt secretion) retain membrane-localized Celsr1, although Celsr1 is present on all faces of node cells. (F,H) Quantification of the intensity of Celsr1 staining shows normal enrichment to the horizontal (anterior and posterior) edges of node cells in DMSO control (F), which is lost when embryos are cultured with IWP2 (H). Scale bars: 10 μm. Data are mean±s.d. ^*P<0.001.

Celsr1 and phospho-myosin light chain 2 are planar polarized in the axial midline. (A,A′) In wild-type embryos cultured for 14-18 hours in 0.1% DMSO, Celsr1 (green) is planar polarized to the anterior/posterior faces in the apical membrane of cells in the midline (outlined in white). (B,B′) Membrane enrichment of Celsr1 is lost when wild-type embryos are cultured in the presence of 10 nM jasplakinolide. (C,C′) In the midline of wild-type embryos, phospho-myosin light chain 2 (green) is also planar polarized at the apical membrane to the anterior/posterior faces of the cell. Scale bars: 10 μm.