Fig. 2. Irx3 provides the posterior competence for Fgf8. Distribution patterns of Irx3 mRNA (A) and its protein (B) in HH21 embryo. (C) Two-color in situ hybridization for Irx3 (purple) and Shh (red) in an HH19 dissected brain. The anterior border of the Irx3-expressing domain is delineated by the ZLI in which Shh is expressed (arrows in A-C). HH16-21 dissected brains were immunostained for En2 (D,G,H-K) or hybridized for Fgf8 (F,L). Electroporation was done at HH8 with Irx3 (D-F), Irx3 and constitutively active Fgfr1 (G), dominant-negative Fgfr3 (H), Irx3 and dominant-negative Fgfr3 (I), Pax2 (J,L) and Pax2 with dominant-negative Fgfr3 (K). The expression vectors for the dominant-negative Fgfr3 and Irx3 or Pax2 were mixed at 9:1 prior to injection. H is an oblique posterior view of the specimen, and the dorsal midline is indicated by dashed lines in H and K, highlighting that the right side (electroporated) of the midbrain region has shrunk and the expression of En2 is severely downregulated. Note that ectopic En2 expression (arrowheads in D) is found only in the vicinity of the Fgf8-expressing sites (arrowheads in F), despite broad expression of the transgenes as visualized by GFP fluorescence (E). Bars, 0.5 mm.