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Fig. 2 MZptk7audm400 mutants show more apoptotic cells prior to notochord vacuolation, leading to a significantly reduced cell number after the vacuolation process. (A) Representative multiphoton images from time-lapse recording of notochord cells from 12-14 hpf until 36 hpf in MZptk7audm400 (right) and their ptk7audm400/+ siblings (left). Various time points (12, 16, 20 and 36 hpf) with GFP staining (top), cell apoptosis staining (middle) and merged (bottom) are shown. Dashed lines mark the boundaries of the notochord. Arrows and arrowhead indicate apoptotic cells and cell debris, respectively. Scale bars: 40 µm. (B) The number of apoptotic cells is indicated at various time points during time-lapse imaging at the region from s1 to s8 or s10. This number is significantly larger in MZptk7audm400 embryos (n=7) compared with their ptk7audm400/+ siblings (n=5) at all stages analyzed. (C) The number of apoptotic cells at the region from s10 to the tail end at 18 hpf, 20 hpf and 22 hpf in MZptk7audm400 embryos (n=7, 14 and 14, respectively) is similar to that of their ptk7audm400/+ siblings (n=8, 10 and 10, respectively). (D) Lateral (top) and dorsal (bottom) views of notochord 3D reconstructions at 5 dpf. Scale bars: 300 µm. (E,F) The total number of vacuolated cells (E) is significantly reduced, and the volume of individual vacuolated cells (F) is significantly larger in MZptk7audm400 mutants (n=4) compared with their ptk7audm400/+ siblings (n=4) at 5 dpf. For B,C,E, P-values were calculated using two-tailed Mann?Whitney U-test. **P<0.01; n.s., not significant. For F, P-value was calculated using two-tailed Welch's t-test.