Figure 3

Nitric oxide overexpression rescues thalidomide affected embryos.

(A–D) Zebrafish eggs (2 cell stages) were electroporated with eNOS GFP plasmid or S1179D. The live embryos (10 hpf) (n = 20 fishes) were grown in presence and absence of 2 mg/ml thalidomide containing water. After 80 hpf the GFP expression was observed under the fluorescence microscope. The pectoral fins abnormalities are presented in scrambled vector control, GFP alone and eNOS GFP electroporated fishes (E–H). Zebrafish eggs electroporated with S1179D were treated with NO specific dye, DAR-4M-AM. The NO production in thalidomide treated or untreated fishes were visualized with the fluorescence microscope. Arrows indicate the pectoral fins. (I) The graphs represent relative intensities of GFP expression and NO production calculated from the images using histogram analysis of image J software. NTF = Non transfected fish; TF = Transfected fish; Thal = Thalidomide treated fish; Thal+Tf = Thalidomide treated transfected fish. *p<0.01 vs eNOS GFP; #p = 0.01 vs S1179D. Inset shows eNOS S1179D expression in transfected zebrafish embryos confirmed using reverse transcriptase PCR. (J) 2 cell stage zebrafish eggs were treated with eNOS inhibitor, L-NAME (10 μM). After 72 hpf the fish embryos were scored for pectoral fin abnormalities. *p<0.05 vs Thal.