Fig. 6. Effects of Lmx1b misexpression on downstream gene expression. (A-F) Effects on Otx2 expression. In situ hybridization for Otx2 (blue) and Lmx1b (red) on the same embryo at 12 hours (A-C') and 24 hours (D-F) after electroporation. (B',C') High-power magnification of boxed areas in B,C. Otx2 is induced by Lmx1b misexpression, but only weakly induced in the isthmus at 24 hours after electroporation (E,F, arrows). (G-I) Effects of Otx2 misexpression on Fgf8 expression. In situ hybridization for Fgf8 (blue) and Otx2 (red) at 12 hours after Otx2 misexpression. Repression of Fgf8 is hardly observed at 12 hours after electroporation (H). (J-M) Effects of Lmx1b-EnR misexpression on Fgf8 expression. In situ hybridization for Fgf8 (J, control; K, experimental side). (L) Section of the specimen shown in J and K, in situ hybridization for Fgf8 (blue) and immunohistochemical staining for HA tag (tagged to Lmx1b-EnR). (M) High power magnification of area in L. As Lmx1b is revealed by immunohistochemical staining, it is localized in the nucleus. Fgf8 signal is localized in the cytoplasm. Fgf8 is induced in Lmx1b-EnR misexpressing cells (M) in the caudal mesencephalon (K,L). (N-P) Effects of co-electroporation of Lmx1b-EnR and Lmx1b on Fgf8 expression. In situ hybridization for Fgf8 at 24 hours after electroporation (blue, N,O). (P) In situ hybridization for Fgf8 (blue) and immunohistochemical staining for HA tag (tagged to Lmx1b-EnR). Ectopic expression of Fgf8 in the caudal mesencephalon is canceled by co-electroporation with wild-type Lmx1b. (Q-T) Effects of Lmx1b misexpression on Grg4 expression. In situ hybridization for Grg4 (blue) (Q,R), and immunostaining for HA (tagged to Lmx1b, brown) (S) at 6 hours after electroporation of pMiw-Lmx1b. Grg4 is induced in the metencephalon on the experimental side (R,S). (T) High-power magnification of the metencephalic region to show that Grg4 is induced in the Lmx1b-misexpressed cells. (A,D,G,J,N,Q) Views from the control side. (B,C,E,F,H,I,K-M,O,P,R-T) Views from the experimental side. Scale bars: 250 µm in F; 200 µm in C,I,K,L,P; 100 µm in S; 25 µm in C'; 10 µm in M,T.

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