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Figure 4 cont. Bad-mediated PCD can regulate cell movement and cells in three-germ-layer targeting to destination sites, as shown via in situ hybridization. Morphological analysis of embryos injected with either 25 ng of control-MO (Bad-M5) or Bad-MO and examined at 8 hpf after ectoderm, mesoderm, and endoderm tissues were stained with cyp26, ntla, and sox17, respectively, via in situ hybridization. (A) The embryos are stained with cyp26 (side view, panels a–c; top view, panels d–f), with the presumptive brain (PB) indicated by white arrows and the mesoderm by red arrows. (B) Quantification of loss-of-Bad-induced PCD effects on ectoderm migration in connection to abnormal brain development at 24 hpf. All data were analyzed using either paired or unpaired Student’s t-tests, as appropriate. p < 0.01. (C) Bad knockdown can induce mild mesoderm defects, as shown by probing with the mesoderm marker ntla gene. Panels a (Bad-M5 group) and b (Bad-MO group): notochord profile indicated by red arrows; margin pattern indicated by white arrow; and forerunner cell indicated by red circle. Bars indicate 100 μm. (D) Quantification of mild loss-of-Bad-induced PCD effects on mesoderm pattern. All data were analyzed using either paired or unpaired Student’s t-tests, as appropriate. p < 0.01. (E) Bad knockdown can induce very mild endoderm defects, as shown by probing with the endoderm marker sox17 gene. Panels a,b, endoderm signal indicated by red arrows and forerunner cell by white arrow. The defect ratio was counted and is shown in (F). All data were analyzed using either paired or unpaired Student’s t-tests as appropriate. p < 0.01. (G) Sketch illustrating how loss-of-Bad-mediated PCD affects smooth cell migration during the formation of the three germ layers at 8 hpf: environmental stress in the form of ROS production was increased, and enhanced cell death further interrupted brain development at a later developmental stage.