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Fig. 3. Targeted disruption of FM enhancer. (A) Wild-type Otx2 allele, targeting vector and recombinant allele. The black box indicates the ApaI-HindIIII 1.4 kp region (FM) to be replaced with a neomycin-resistant gene (Neo, open boxes) flanked by loxP sequences (black triangles). DT-A is the diphtheria toxin A fragment gene with MC1 promoter, which is used for the negative selection of homologous recombinants (Matsuo et al., 1995). Thick and thin lines indicate genomic and vector-derived sequences, respectively. Probe A is the Southern blotting probe employed for identification of homologous recombinant ES cells displayed in the right panel. (B) FM enhancer mutant phenotype. Wild-type (a,b), Otx2^FM/FM (c,d) and Otx2^FM/– (e,f) embryos at E12.5 (a,c,e) and E15.5 (b,d,f). A double arrowhead in f indicates the expanded cerebellum primordium. The phenotypes were examined with both the Otx2^FM mutant in which the neo insert remained and the Otx2^FM mutant in which the insert was deleted by Cre recombination. No differences were found, and the following marker analyses were performed with the mutant that retained the neo insert. (C) Marker analysis of Otx2^FM/– phenotype. Otx2 (a,b), Pax6 (c,d), Fgf8 (e,f) and Emx2 (g,h) expression in E10.5 wild-type (a,c,e,g) and Otx2^FM/– (b,d,f,h) embryos. Scale bars: 400 µm.

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