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Fig. 1. Generation of the tOtx2^bov mutant and Otx2 overexpression in ES cells and mutant embryos. (A) The genomic position at the chromosome 7 D2 region (upper line) where the tOtx2^bov cassette (second line) is inserted allows the genotyping of mutant embryos by using two pairs of primers (open arrows in the upper line for the wild type and open arrows in the second line for the mutant). Cre-mediated removal of the Neo-triple polyA stop cassette generates the tOtx2^ov allele (third line) and this event is monitored by the primers shown in the second and third line (black arrows). (B) The R26^CreER/+; tOtx2^bov/+ ES clone is treated with tamoxifen for 24 hours prior to monitoring the removal of the Neo-triple polyA cassette (upper panel), the Otx2 protein level (second panel) and the GFP activation (third panel). (C-K) Immunohistochemistry and in situ hybridization performed to detect the expression of Otx2 (C-E), En1-driven Cre recombinase (F-H) and GFP transcripts (I-K) in adjacent sagittal sections of E10.5 En1^Cre/+, En1^Cre/+; tOtx2^ov/+ and En1^Cre/+; tOtx2^ov/ov embryos show that Otx2 is ectopically activated in the anterior hindbrain and cerebellum anlage (arrows in D,E) and, as revealed by GFP expression, overexpressed in the whole mesencephalon (J,K). Abbreviations: Mes, mesencephalon; Hb, hindbrain; MHB, midbrain-hindbrain border.

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