Fig. 4. En1^cre/+; Otx2^flox/flox; Otx1^-/- triple mutants show anterior shift of ventral expression of Fgf8. (A-E) In situ hybridisation on E10.5 En1^cre/+; Otx2^flox/flox; Otx1^-/- whole mount embryos and sections with Otx2 (A), Fgf8 (B), Gbx2 (C), lacZ (D) and Otx2-5' (E) probes. The sections are focussed on the ventral midbrain area approximately corresponding to the area included in the dotted square in B. The arrows in A,D,E indicate the anterior border of the Fgf8 domain and the arrows in B indicate the ventral posterior border of the functional Otx2 expression domain. Compared with En1^cre/+; Otx2^flox/flox embryos, the expression domains of Fgf8 and functional Otx2 are adjacent, while posterior to Fgf8, the presumptive midbrain yet exhibits transcription of Otx1 (lacZ) and Otx2 (Otx2-5'). Hb, hindbrain.