Fig. 5

Dvl2-EGFP was primarily localized to the membrane in the neuroepithelium during neurulation. Single optical sections from laser-scanning confocal microscopy of neuroepithelium from neurulating embryos showed that, in the wild-type background, Dvl2-EGFP was primarily localized to the plasma membrane in the neuroepithelium (A,E,I). Consistent results were observed in wild-type and Lp/Lp mutant two-somite stage embryos stained with an anti-EGFP antibody (A,B) or through direct visualization of Dvl2-GFP signal from briefly fixed five-somite stage embryo (E,F) or live eight-somite stage embryos in culture (I,J). The apparently diffuse cytoplasmic distribution of Dvl2-EGFP and actin in some cells in A,B was due to scanning of the focal plane immediately underneath the apical plasma membrane during confocal laser microscopy. Wild-type and mutant embryos from the two- and five-somite stage were also stained with Phalloidin (C,D,G,H) to label F-actin. When the entire series of z stacks were examined (data not shown), it was clear that both Dvl and actin are localized to the lateral plasma membrane as individual optical sections are scanned more basally through the cell. Curiously, Dvl2-EGFP and actin distribution also appeared to be more diffuse in the somatic mesoderm at the five-somite stage (E-H). Overall Dvl2-EGFP distribution in the neuroepithelium was not altered in the Lp/Lp mutants (B,D,F,H,J,L; data not shown). (K,L) DIC images of live wild-type (K) or Lp/Lp mutant (L) embryos in culture. NE, neuroepithelium; SM, somatic mesoderm.