Fig. 1. Pattern of expression of Fgf8 in developing diencephalon. The spatial distribution of Fgf8 and its activity are shown by whole-mount in situ hybridization at E9.5 in mice. Strong expression of Fgf8 is observed at the midbrain/hindbrain boundary (MHB, red arrow) and in the anterior telencephalon (black arrow) (A). Strong expression of Shh is observed in the basal region of the diencephalon (black arrow) (B). (C-F) Whole-mount in situ hybridization of the dissected brain at E10.5 is shown. Lateral is towards the left. In addition to Fgf8 expression in MHB and anterior telencephalon (red arrow and black arrow, respectively), expression in the dorsal part of diencephalon (green arrow) and dorsal to ZLI (orange arrow) is detected. One side of the hemisphere has been removed for a better view of Fgf8 expression in the diencephalon (C). Shh expression in ZLI is shown (blue arrow) (D). Two-color in situ hybridization for Fgf8 (blue) and Shh (brown) shows the spatial relationships of Fgf8 and Shh expression (arrow) (E). FGF activity, shown by Sprouty2 (Spry2, blue), is detected in both p2 and p3 (arrows), which are divided by Shh (brown) (F). (G-I) Section in situ hybridization shows the detailed patterns of expression of Fgf8 and Shh. Estimated section positions are shown in E and F (broken line). Fgf8 expression is restricted to p3 (arrow) (G) and a high-magnification view is shown (I). Fgf8 activity shown by Spry2 reached a greater distance on both sides of ZLI marked by Shh (H) at E10.5. di, diencephalon; H, hindbrain; M, midbrain; tel, telencephalon. Scale bar in F: 500 µm for A-F; 200 µm for G,H; 100 µm for I.