Ceramide generation through thalidomide-activated nSMase in zebrafish embryos, and restoration of thalidomide-induced antiangiogenic action by antisense-morpholino oligonucleotides (MOs) for nSMase. (A-C) Zebrafish embryos were treated at 75% epiboly stage (8 hpf) with the indicated concentrations of thalidomide in the presence or absence of antisense- or invert-MO for nSMase, and harvested at 18 hours after treatment (26 hpf). Thalidomide-induced increase of ceramide generation through nSMase activity peaked at 12 to 18 hours after treatment (A). Injection of antisense-MO for nSMase into embryos inhibits thalidomide-increased activation of nSMase (B) and ceramide generation (C), but that of invert-MO does not. (B-C) ▪ indicates presence of thalidomide; ▪, absence of thalidomide. The results are obtained from 3 independent experiments. The error bars indicate 1 SD. *Statistical significance of antisense-MO effect as compared with invert-MO effect (P < .01). (D,E) Hematoxylin and eosin staining for the transverse section of antisense-MO–injected 48-hpf embryos shows the restoration of the dorsal artery (marked “A”) and posterior cardinal vein (marked “V”) against thalidomide-induced defects (original magnification, ×150). (F-I) Lateral view of in situ mRNA hybridization for FLK-1 at 30 hpf shows that the injection of antisense-MO rescues thalidomide-inhibited FLK-1 expression, as indicated by a rectangular shape (original magnification, ×40; F,H). The restoration by antisense MO of thalidomide-inhibited FLK-1 expression is more evident by the higher magnification (original magnification, ×63; G,I). The results are representative of each experiment (D-J). noto indicates notochord; A, dorsal artery; V, posterior cardinal vein; hpf, hours after fertilization; MO, morpholino oligonucleotide; nSMase, neutral sphingomylinese. Image acquisition was performed as described for Figure 1.