Delayed closure revealed by quantification of closure speed from live-imaging data. (A) Measurement of L (length of MHNP). Rostral and caudal edges of MHNP were marked in 3D data (red circles), and their coordinate values were used for calculation of L. (B) Representative images of MHNP closure in control (apaf-1^+/+ or apaf-1^+/−, apaf-1^−/−, and zVAD-treated embryos). t represents the imaging duration (minutes). (A and B) Bars, 100 µm. (C and E) Kinetic analysis of NTC in zVAD-treated embryos (C57BL6×ICR F1 offspring; control, n = 6; zVAD treated, n = 5; C) or in mutant embryos (C57BL6;129S1 mixed background; apaf-1^+/+ or apaf-1^+/−, n = 11; apaf-1^−/−, n = 4; E) and length of the MHNP plotted against time. Closure speed was calculated from the linear approximations shown in the graphs (black solid lines and red dashed lines). Note that the closure speed in each embryo was almost constant during the culture period. (D and F) Closure speeds quantified in C and E were significantly slower under caspase-inhibited conditions than under control conditions (*, P < 0.05).