Regional preferences in caspase activation between C- and D-type apoptotic cells. Immunostaining of active caspase-3 (a-Casp3) or active caspase-7 (a-Casp7) in embryos undergoing the cranial NTC. Magenta represents Hoechst 33342 staining. (A) In D-type cells, nuclear fragmentation and pyknotic nucleus were observed (magenta; arrows). Bar, 100 µm. (insets) Magnified images of the boxed area are shown. Bar, 10 µm. (B) Active caspase-3 staining, which looked like fragmented apoptotic bodies, was observed in the dorsal ridge of neural plate (green; arrowheads). Bars: (top left) 100 µm; (top right) 50 µm; (bottom left) 10 µm. (C) Signal of cleaved lamin A, which was a substrate of activated caspase, was detected in the D-type cell (green; arrow). Bar, 25 µm. (insets) Magnified images of the boxed area are shown. Bar, 10 µm. (D) Active caspase-3 staining was detected in the surface ectoderm adjacent to the boundary cells (green; arrowheads). It was also observed in rounded D-type cells at the zipping regions (green; arrows) and in the cells aligning the dorsal ridge of the neural plate (green; asterisks). Bars, 25 µm. (E) Active caspase-7 was detected in the D-type cells (green; arrows) detached from the boundary domain. Their nucleus was pyknotic (magenta), indicating that the cells were dying. Bars: (left) 100 µm; (middle) 25 µm; (right) 10 µm. (F) Distribution of two types of apoptotic cells during NTC. The neural plate (NP) is shown in purple, the surface ectoderm (SE) in green, and the boundary cells (BC) between the neural plate and surface ectoderm in orange.