EGFP and CYP3A7 expression in hCYP3A7/EGFP-expressing zebrafish. A pT2A plasmid containing hCYP3A7/EGFP (3A7: C,D,G,H,J–L) was injected together with transposase cmRNA into 1-cell stage embryos. (A–D): 17 hpf, (E–H): 50 hpf. EGFP fluorescence in wild-type (WT) zebrafish (B,F) and that in hCYP3A7-expressing zebrafish (hCYP3A7 zebrafish, 3A7) (C,D,H) are shown. Image (A) is a bright field image of a wild-type embryo. Images (E,G) are bright field images of (F,H), respectively. CYP3A7 mRNA expression was observed at 23 hpf by WISH (I–L). Arrowheads indicate examples of positive signals in J–L. Scale bars = 200 µm.

Effects of thalidomide on pectoral fin development of hCYP3A7-expressing zebrafish. Embryos/larvae of wild-type (WT) zebrafish (A,B) and hCYP3A7-expressing zebrafish (3A7) (C–G) were exposed to 200 μM thalidomide (TD) or 0.02% DMSO as a vehicle from 17 hpf to 72 hpf. Arrows in (D–G) indicate pectoral fin malformation. The white arrowhead in (F) indicates bleeding in the dorsal head region. Black arrowheads show a smaller eye (F) and loss of an eye (G). Scale bar = 200 µm. A summarized graph is presented in H (n = 6, 8–26 zebrafish for each case). * p < 0.05, ** p < 0.01.

Effects of thalidomide on pericardial edema and otic vesicles of hCYP3A7-expressing zebrafish. Embryos/larvae of wild-type (WT) zebrafish (A,B,E,F) and hCYP3A7-expressing zebrafish (3A7) (C,D,G,H) were exposed to 200 μM thalidomide (TD) or 0.02% DMSO as a vehicle from 17 hpf to 72 hpf. The arrow in D indicates severe pericardial edema. The arrow and arrowhead show loss of a large otolith (saccule) and round otic vesicle, respectively. Scale bar = 200 µm. Summarized data are presented as a graph in (I) (n = 4, 8–26 zebrafish for each case). ** p < 0.01.

Effects of thalidomide on hCYP1A1-expressing zebrafish during development. Embryos/larvae of wild-type (WT) zebrafish and hCYP1A1-expressing zebrafish (hCYP1A1-zebrafish) were exposed to 200 μM thalidomide (TD) or 0.02% DMSO as a vehicle from 17 hpf to 72 hpf. (A,B, E,F): wild-type zebrafish. (C,D,G,H): hCYP1A1-zebrafish. Zebrafish in (A,E,C,G) were exposed to DMSO. Zebrafish in (B,F,D,H) were exposed to thalidomide. (A–D): dorsal views; (E–H): lateral views. Scale bars = 200 µm.

Thalidomide-induced reduction in fgf8 expression in pectoral fin buds of hCYP3A7-expressing zebrafish. Wild-type (WT) (A,B) and hCYP3A7-expressing embryos/larvae (CYP3A7) (C,D) were exposed to 200 µM thalidomide (TD) or 0.02% DMSO (DMSO) as a vehicle from 17 hpf until 48 hpf. Larvae were fixed at 48 hpf for whole-mount in situ hybridization with an fgf8 probe. Summarized data are presented as graphs in (E,F) (n = 3, 7–10 embryos for each case). (E,F) indicate percentages of fgf8 expression and percentages of unilateral and bilateral loss of fgf8 expression in the presence or absence of thalidomide. * p < 0.05. Scale bar =100 µm.