Time-lapse imaging of the cranial NTC under a fast-scanning confocal microscope. (A) System for time-lapse imaging of the cranial NTC. Embryos undergoing NTC (somite stage 9–11 for zipping closure I in rhombencephalon and somite stage 12–16 for closures I and II to seal MHNP) were dissected from the uterus and yolk sac and were placed into holes in 2% low temperature–melting agarose on glass-bottom dishes, which were filled with culture medium, and placed in a humidified 37°C/5% CO₂ incubator in order to visualize the hindbrain region (red rectangle). (B, left) Multiple closure sites in the mouse embryo. α and β represent initiation sites of closures I and II, respectively. The MHNP was observed by live imaging (asterisk enclosed by the square). (right) The MHNP (asterisk) is closed by closures I and II. (C) A schematic illustration of the cranial NTC. (i–iii) NTC steps shown as transverse sections along the rostrocaudal axis (indicated in B for ii and iii). (iv) High-magnified view of the dorsal ridge. We termed regions around the boundary cell as the boundary domain (shown by blue circle), including the adjacent surface ectoderm and the edge of the dorsal ridge of the neural plate (red circle). (D) Time-lapse imaging of the cranial closure I in SCAT3 transgenic mice. ot, otic placode. t, imaging duration (minutes). Flipping of the neural ridges in the prospective midbrain and hindbrain is emphasized by dotted lines. Bars, 150 µm. (E) Zipped regions were traced (jagged blue line). Bars, 100 µm. (insets) High-magnification views of zipping are shown. Bars, 25 µm. (D and E) Arrows indicate forward zipping edges of closure I. (F) Time-lapse imaging of closures I and II at the MHNP (asterisks) in SCAT3 transgenic mice. Arrows indicate forward zipping edges of closures I and II. Bars, 50 µm.