RhoGEF3 is required for apical constriction. (A–A″) RhoGEF3-GFP (green) was detected at low levels at apical junctions of the NE (Patj, red; E-cad, magenta) and appeared to be up-regulated in NSCs and epi-NSCs (white arrows); epi-NSCs were identified via the down-regulation of Patj. (B and B″) Down-regulation of Patj (green) appeared to occur normally in cells located at the medial edge of the NE in rhoGEF3 mutant brains (white arrows; E-cad, red). (C and D) Apical constriction of epi-NSCs (Neur-GFP, green in C) was reduced in rhoGEF3 mutant brains (C and C′; E-cad, red), as shown in the quantification (D) of the apical area in rhoGEF3 mutant (n = 9 brains) versus wild-type brains (n = 7; data presented as in Fig. 2 B, with 149–326 cells per binned distance; ***, P = 7 × 10⁻¹⁹, two-tailed Student’s t test). (E and E′) Junctional MyoII (MyoII-TagRFP, red) appeared to be enriched along at cell–cell contacts between epi-NSCs (Neur-GFP, green) and more lateral TZ cells (white arrows). (F) Box plots of MyoII intensity along junctions of the NE based on the four distinct junction identities described in Fig. 3 B, showing that b junctions had a significant enrichment of MyoII in rhoGEF3^KO brains (n = 7 brains, b vs. a, b, or c, ***, P values <10⁻¹², one-way ANOVA, Scheffé multiple comparison tests). Scale bars = 10 µm.