Fig. 4

Knockdown of hace1 and overexpression of rac1-V12 cause a delay in neural tube closure. a Embryos were injected with the indicated MOs (30 ng) into the animal region of two dorsal blastomeres at the four-cell stage and the images were collected at stage 20. Anterior views with dorsal to the top. Representative results from two independent experiments are shown. b HACE1 MO (15 ng) was injected into the animal region of the right dorsal blastomere, and control MO (15 ng) was subsequently injected into that of the left dorsal blastomere. rac1-V12 (constitutively active Rac1) mRNA (10 pg) and gfp mRNA (90 pg) were injected into the animal region of the right dorsal blastomere, and then gfp mRNA alone was injected into the animal region of the left dorsal blastomere. Embryos at stage 13 were placed on a culture dish, and images were collected every 5 min until embryos completed neural tube closure. Still frames from a time-lapse movie show embryos from stage 14 (0 min) to stage 18 (120 min). Dorsal views with anterior to the top. Dotted lines indicate the midline. Transient posterior protrusion (e.g., middle left) was sometimes observed because embryos were firmly embedded in the agarose groove and thus were constantly subjected to the lateral-to-medial force. c A magnified view of embryos in b (60 min). Black dotted lines indicate the inner edges of neural folds. d The maximum distance between the dorsal midline and the left (i) or right (ii) inner edge of the neural fold was measured (shown as white double-headed arrows in c). The differences ((ii) minus (i)) were then calculated and plotted (Uninjected, n = 17; HACE1 MO, n = 20; Rac1-V12, n=11). Long central bar marks mean and upper and lower bars mark s.d. *P < 0.05, **P<0.01, Mann–Whitney U-test