Posterior shift of the IsO in Sp8^-/-embryos. Whole-mount in situ hybridization showing lateral views of E8.5 control (A) and mutant (B) embryos hybridized with Otx2 and dorsal views of Gbx2 expression in control (C) and mutant (D) embryos at a similar stage of gestation. Rhombomeres (r), 3 and 5 are labeled in all embryos with Krox20 transcripts. The distance between the posterior limit of Otx2-expression and the anterior boundary of r3 is indicated by a red line in mutant and control embryos. This line also indicates the distance between the posterior limit of Gbx2-expression in r1 and the anterior boundary of r3.

At E9.5, the expression domains of genes transcribed at the MHB is enlarged. Wholemount in situ hybridization showing the expression of Otx2 (A,B), Gbx2 (C,D), Fgf8 (E,F), En1 (G,H), Pax2 (J,K) and Wnt1 (L,M) and transcribed at the MHB, as indicated. At this stage of development (E9.5), the shift of the IsO, detected at E8.5 and shown in Fig. 2, is still evident (A-D), and the expression domains of the presented markers appear enlarged at the MHB of the mutant embryos (D,F,H,K,M). The embryos shown for Wnt1 are between E9.5 and E10.

At E10.5, the Otx2/Gbx2 expression boundary appears normal but ectopic expression of Fgf8, Otx2 and Wnt1 is found in the ventral part of the rostral hindbrain. The expression of Otx2, Gbx2, Wnt1 and Fgf8 is shown on consecutive sagittal sections of E10.5 control and mutant embryos, as indicated. The embryos in B and D are different from those in F and G. (A-D) Expression of Fgf8 and Wnt1 is presented in consecutive sagittal sections of wild type (A,C) and mutant (B,D) embryos. The expression of Fgf8 appears more affected and therefore more widespread when compared with Wnt1, and ectopic cell patches seem to reach the most posterior midbrain (arrow in B). In the ventral midbrain, the expression of Wnt1 (D) is patchy and more widespread than in the control embryo. (E-H) Expression of Gbx2 and Otx2 on consecutive sagittal sections of wild type (E,G) and mutant embryos (F,H). The ectopic expression of Otx2 and Wnt1 in the rostral hindbrain is found only in the ventral part, while for Fgf8 this is also true for the dorsal part. (J,K) Expression of Pitx3, as a marker for midbrain dopaminergic neurons (Smidt et al., 2004), on sagittal sections of E12.5 embryos. Ectopic Pitx3 signal can be found in the ventral part of the rostral hindbrain of Sp8-deficient embryos, arrowhead in K. (L-O) Expression of ephrin A5 as a marker for the inferior colliculus (Donoghue et al., 1996) was not modified at E11.5 of gestation, as detected on sagittal sections of two mutant embryos (M,O). Ephrin A5 expression is, however, upregulated in the ventricular zone of the ventral midbrain of these mutants (M,O, arrows) when compared with wild type embryos (L,N).

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).

The loss of Sp8 provoked overgrowth of the neuroepithelium. (A-M) Expression analysis of several markers of the ventral midbrain: Shh (A,B), Nkx6.1 (C,D), Nkx2.2 (E,F), Wnt5a (G,H), Wnt1 (J,K) and Foxa2 (L,M) on frontal vibratome sections (45 μm) selected at the level of the posterior midbrain. The expression pattern of all these markers presented here reflects the expansion of the neuroepithelium and does not reveal a patterning defect at this stage of development. The overgrowth of the neuroepithelium results in reduced aqueduct space.

Enhanced cell proliferation in the mid- and rostral hindbrain of Sp8-deficient embryos. (A-D) BrdU-labeled cells with a pulse of 30 minutes are shown in frontal sections, at the level of the posterior midbrain (A,B) and in sagittal hindbrain sections (C,D) of 10.5 wild-type and mutant embryos. In mutant embryos, a remarkable increase in the number of proliferating cells was evident, thus suggesting that Sp8 negatively regulates cell proliferation. All images are at the same magnification. A and B are 5 μm sections; C and D are 10 μm sections.

Cell differentiation may be delayed at E11.5 in the absence of Sp8. (A-D) Double-immunohistochemical analysis presenting the expression of nestin (green) and β-III-tubulin (Tuj1, red) as markers labeling undifferentiated neuronal progenitors and differentiating neurons, respectively. In Sp8 mutant embryos (E11.5), as indicated by the arrowheads in B and D, there are some cells outside of the ventricular zone that still express nestin but are devoid of Tuj1 signal. This suggests that these cells might correspond to neuronal progenitors that are not fully differentiated. C,D are at higher magnification than A,B.