Signaling through ROCK is required for ephrin-B1–mediated cell segregation in mouse embryos. (A, D, and G) Comparison of patches resulting from aberrant cell segregation in the frontonasal processes of E11.5 Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+ embryos. Sections were stained for ephrin-B1 (magenta) and GFP (green), and GFP-positive cells and patches in the lateral nasal process (cropped images) were quantified and are outlined in yellow for clarity. Bars, 100 µm. (A) An unsorted embryo (Efnb1^loxXGFP/+) compared with an Efnb1 heterozygous embryo (Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;ROCKDN^+/+) and an Efnb1 heterozygous embryo with Wnt1Cre2-induced expression of dominant-negative ROCK (Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;ROCKDN^lox/+). (D) Control heterozygous embryo (Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Cdc42^lox/+) compared with a heterozygous embryo with loss of Cdc42 (Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Cdc42^lox/lox). (G) Control heterozygous embryo (Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Rac1^lox/+) compared with a heterozygous embryo with loss of Rac1 (Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Rac1^lox/lox). (B, E, and H) Distribution of GFP-positive areas into patches of various sizes. Column heights represent means of the distributions in each embryo, and error bars represent SEM. ****, P < 0.0001 as compared with Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+. (C, F, and I) Patch sizes represented as scatterplots. Horizontal bars represent means, and error bars represent SEM. *, P < 0.05; ***, P < 0.001, compared with Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+; ****, P < 0.0001 compared with Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Rac1^lox/+ control group. Red symbols, Efnb1^loxXGFP/+. Blue symbols, Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;ROCKDN^+/+, Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Rac1^lox/+, or Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Cdc42^lox/+. Green symbols, Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;ROCKDN^lox/+, Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Rac1^lox/lox, or Wnt1Cre2^Tg/+;Efnb1^loxXGFP/+;Cdc42^lox/lox. Number of embryos analyzed are presented in Table S2.