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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.
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