S1P prevents thalidomide-induced antiangiogenic action by inhibition of ceramide generation through nSMase. (A-C) Zebrafish embryos at 60% epiboly stage (7 hpf) were pretreated with the indicated concentrations of S1P for 1 hour before treatment with 800 μM thalidomide, and harvested at the indicated times (A) or 18 hours after treatment (B, C). (A) Thalidomide-induced generation of ceramide (•), peaked 18 hours after treatment, is decreased by pretreatment with S1P (▪). S1P dose-dependently inhibits thalidomide-induced generation of ceramide and activation of nSMase (B,C). The results are obtained from 3 independent experiments. The error bars indicate 1 SD. *Statistically significant inhibition by 0.5 μM S1P of 800 μM thalidomide-induced effects (P < .01). (D,E) Lateral view of a fixed embryo at 30 hpf shows that pretreatment with S1P restores thalidomide-caused shortness of embryo (white arrows). (F,G) Hematoxylin and eosin staining for the transverse section shows that thalidomide-induced defects of the dorsal artery (marked “A”) and vein (marked “V”) are reconstructed by pretreatment with S1P. (H-M) In situ mRNA hybridization was performed for endothelial markers neuropilin-1 (H,I) and FLK-1 (J-M). Thalidomide-caused inhibition of neuropilin-1 and FLK-1 expressions at the large vascular region is restored by pretreatment with S1P, as indicated by boxes (I,L). The restoration by S1P of thalidomide-inhibited FLK-1 expression at large vessels is more evident by the higher magnification (K,M). The results are representative of each experiment. noto indicates notochord; A, dorsal artery; V, posterior cardinal vein; hpf, hours after fertilization. Original magnifications of panels are as follows: D, E, H, I, J, L, ×40; F, G, ×150; and K and M, ×63. Image acquisition was performed as described for Figure 1.