Fig. 3. Otx2 overexpression induces AP graded increase of mdDA neurons. (A-L'') Immunohistochemistry performed at six sequential anatomical levels corresponding to the posterior pretectum and anterior mesencephalon (A-B'',G-H''), the intermediate mesencephalon (C-D'',I-J'') and the posterior mesencephalon (E-F'',K-L'') of E12.5 wild-type, En1^Cre/+; tOtx2^ov/+ and En1^Cre/+; tOtx2^ov/ov embryos with Pitx3 and Nkx6.1 (A-F'') and Otx2 and Nkx2.2 (G-L'') shows that in Otx2-overexpressing embryos the number of Pitx3⁺ neurons gradually increases, moving from the anterior towards the posterior mesencephalon. This increase correlates with the copy number of the Otx2-overexpressing allele. Conversely, the Otx2 overexpression does not affect the extent of progenitor domains located dorsal to the mdDA domain; indeed, while the Nkx6.1^--Nkx2.2^- mdDA domain (ventral to the arrow) gradually expands, the Nkx6.1⁺-Nkx2.2^- (between arrow and arrowhead) and the Nkx6.1⁺-Nkx2.2⁺ (dorsal to the arrowhead) domains retain a similar distribution among the three genotypes at the different anatomical levels. Abbreviation: Ant, anterior.