Figure 6. CRB2 regulates the distribution of Myosin IIB.

(A) En face view of the apical surface of the epiblast layer of the primitive streak at E8.0, showing the reciprocal enrichment of CRB2 and Myosin IIB on different cell edges; proximal is up. (A) Single optical section of whole mount immunostaining for CRB2 (red) and Myosin IIB (green). (B) Magnification of boxed region in A, with single channels for CRB2 and Myosin. (C) Quantification of Myosin IIB and CRB2 en face localization at cell edges at the primitive streak (see Methods). Cell edges with high CRB have low levels of Myosin IIB (normalized intensity of Myosin IIB at high Myosin edges = 1.48 (n=349 cell edges) and normalized intensity of Myosin IIB at high CRB2 edges = 0.6 (n=436 cell edges); two tailed student t-test, p<0.00001, n=5 wild-type embryos). Edges with high Myosin IIB have low levels of CRB2 (normalized intensity of CRB2 at high Myosin edges = 0.57 (n=349 cell edges) and normalized intensity of CRB2 at high CRB2 edges = 1.36 (n=436 cell edges); two tailed student t-test, p <0.00001. The box represents the 25^th–75^th percentile, whiskers indicate 1.5 times the range, dots are the outliers and bar in the middle is median. (D–E) Myosin IIB along the cell edges is reduced in Crumbs2 mutants. (D) Extended projection view of the primitive streak of embryos whole mount stained for Myosin IIB and phalloidin at E8.0 showing lower levels on Myosin IIB on the cell edges in Crumbs2 compared to wild-type embryos. (E) Quantification of relative staining intensity shows that the total level of Myosin IIB on cell edges is ~2 fold higher in wild type than in Crb2 mutants (WT mean myosin intensity: 17.25×10³, n=570 edges, 4 wild-type embryos; mutant mean myosin intensity: 9.32×10³; n= 570 edges, 3 mutant embryos, two tailed student t-test, p <0.00001). The box represents the 25^th–75^th percentile, whiskers indicate 1.5 times the range, dots are the outliers and bar in the middle is median. Scale bars A and B, 10 μm; D, 20 μm. (F) Model for actions of CRB2. CRB2 controls E-cadherin down-regulation by promoting the SOX2-to-SNAIL1 switch, and controls Myosin IIB accumulation through a second pathway, perhaps through aPKC and Rho kinase. The complex anisotropic patterns of CRB2 and Myosin IIB are regulated in part by self-organizing regulatory mechanisms. The images shown represent the mutant and wild type from the same experiment.