Real-time detection of apoptotic cells during NTC. Caspase activation is indicated by the V/C (1.0–0.5). (A–C) Concomitant occurrence of apoptosis and morphological changes in the boundary domain. t is indicated in minutes. (B) High-magnification view of insets in A. (i and ii) V/C (i) and ECFP (ii) images. The boundary cells and surface ectoderm (SE) are colored yellow in ii. Concomitant with the flipping of the dorsal ridge of the neural plate (NP), apoptotic cells (blue) appeared beneath the flipping ridges. (iii) Reconstituted transverse (XZ) sections from 4D datasets at r2, indicated by yellow lines in i (also in A). The flipping of the dorsal ridges and bending of the neural plate are indicated by dotted lines. (C) High-magnification views of the flipping dorsal ridge in A and B. Apoptotic cells danced around the dorsal ridge and tumbled down the neural plate (arrows) or disappeared (arrowheads). The flipping edge of the neural plate was being covered by the boundary cells and surface ectoderm, which are colored yellow in the ECFP images (as in B, ii). (D) Features of D- and C-type apoptosis during cranial NTC. (E) A cell showing activated caspase (arrows in V/C images; t = 260) shrank (t = 260) and became fragmented (t = 276; arrowheads) in the boundary domain before the completion of NTC. The same cell is circled by magenta in the ECFP images in the magnified images of the boxed areas. (F) After the MHNP was sealed, extensive caspase activation (arrows) and subsequent cell fragmentation (arrowheads) occurred at the midline. The same cells are circled by magenta in the ECFP images. Bars: (A) 100 µm; (B and E) 50 µm; (C and F) 25 µm; (D) 6 µm.