Figure 3.
A, Correct positioning and maintenance of the isthmic organizer. Whole-mount in situ hybridization of brains from control and Otx2-CKO embryos at E12.5 for Otx2 (A, A′), Fgf8 (B, B′), Wnt1 (C, C′), and Gbx2 (D, D′). All genes show identical patterns of expression in mutant and control embryos, indicating that the mid-hindbrain boundary organizer is correctly positioned and maintained in the Otx2-CKO mutants. Arrowheads mark the position of the isthmus. cer, Cerebellum; mes, mesencephalon; rh, rhombencephalon. Scale bars: (in A) A, A′, B, B′, D, D′, and (in C) C, C′, 200 μm. Anterior is to the top. B, D-V patterning in the midbrain of Otx2-CKO embryos. In situ hybridization of coronal sections at the level of the midbrain DA field at E12.5. Shh expression is similar in control and Otx2-CKO mutant embryos (compare E, E′) with the exception of its most dorsal expression, in which the intensity of expression is decreased (brackets in insets in E and E′). Ptch1 (F, F′) and Gli1 (G, G′) show no changes in their expression, and their dorsal limit of expression, marked by arrowheads, is identical in control and mutant embryos. Moreover, Pax3 expression is not affected in Otx2-CKO compared with control embryos (arrowheads in H and H′ indicate ventral boundary of expression of Pax3). Black arrows indicate the position of the alar-basal boundary (E-H, E′-H′). Gfd7 is also properly expressed in Otx2-CKO mutant embryos (I, I′). In contrast, Msx1 expression is slightly expanded in the roof plate of Otx2-CKO mutants (J, J′). Msx1 is also expressed in the surface ectoderm of Otx2-CKO mutants like in control embryos (Fig. 3 J, J′). Scale bars: (in E) E, E′, F, F′, G, G′, H, H′, 200 μm; (in I) I, I′, J, J′, 50 μm.