Fig. 5. Otx2 and/or Gbx2 are not required for the activation but for the restriction of Sp8 expression at the MHB. The expression of Sp8 is shown, where possible, at different stages of development in wild-type (A,E,H), Gbx2^-/- (B,F,I), Otx1²/Otx1² (C,G) and Otx1²/Otx1²/Gbx2^-/- (D) embryos. In situ hybridization was performed on sagittal sections and using ³⁵S-labeled Sp8 riboprobe. Although the Otx1²/Otx1²/Gbx2^-/- double mutant is lethal between E9.2 and E.9.5 and therefore cannot be presented at E10.5 and E12.5, the in situ with Otx1²/Otx1² is shown at E10.5 as the E12.5 phenotype is essentially the same as at E10.5. As can be seen, Sp8 is not downregulated in embryos lacking Gbx2 (B,F,I), Otx2 (C,G) or both proteins (D). Sp8 is rather strongly expressed in Gbx2-deficient embryos in the rostral hindbrain committed to be transformed in an expanded posterior midbrain (Wassarman et al., 1997) (B,F,I). In Otx1²/Otx1², the rostral tip of the central nervous system should correspond to an isthmus-like structure (Martinez-Barbera et al., 2001) and Sp8 is expressed here along all the CNS (C,G). In Otx1²/Otx1²/Gbx2^-/- double mutant, Sp8 is expressed along all the anterior neural plate (D). In this double mutant, this territory fails to activate forebrain- and midbrain-specific markers, while it shows ubiquitous expression of all the genes transcribed at the MHB (Martinez-Barbera et al., 2001) and is therefore considered as an expanded MHB. Thus, Sp8 expression is similar to what has been reported previously for other gene functions transcribed at the MHB (Martinez-Barbera et al., 2001; Li and Joyner, 2001).