Fig. 7. Distinct disruption of Fgf8a and Fgf8b by siRNA. (A) Alignment of partial sequence of Fgf8a and Fgf8b mRNA. The target sequence of Fgf8a- and Fgf8b-siRNA is underlined with red and green, respectively. The number indicates the number from the start codon. (B-E) Effects of Fgf8b-siRNA on Fgf8 expression. We could detect disruption of Fgf8 mRNA to some extent. Since the probe used for in-situ hybridization hybridized to both Fgf8a and Fgf8b, disruption of Fgf8b may be more than we can detect. (F-I) Effects of Fgf8a-siRNA on Fgf8 expression. We could not detect the effect of siRNA. This may be due to the fact that Fgf8b is predominantly expressed in the isthmus. (J-M) Application of both Fgf8a- and Fgf8bsiRNA. Electroporation of both Fgf8a- and Fgf8b-siRNA resulted in distinct reduction of Fgf8 mRNA in the isthmus. (N-P) Effects of siRNA on the activation of ERK. Fgf8b-siRNA decreased the activation level of ERK (N), although Fgf8a-siRNA hardly affected ERK activity (O). (Q-S) Effects of Fgf8b-siRNA on Otx2 expression. The arrows represent the caudal border of the Otx2 expression domain. Application of Fgf8b-siRNA by electroporation resulted in a caudal shift of the Otx2 expression domain; in other words, the mesencephalon extended caudally. (T-V) Effects of Fgf8a-siRNA on Otx2 expression. Fgf8a-siRNA did not affect Otx2 expression. Dorsal view (B,F,J,N-Q,T), Lateral view (C,D,G,H,K,L,R,U), GFP fluorescence to indicate the site of siRNA introduction (E,I,M,S,V). mes, mesencephalon; met, metencephalon; cont, control side; exp, experimental side. Scale bars: 200 µm.