Fig. 3. Expression of mes-met genes in single Otx2 or Gbx2 and double homozygous mutant embryos at early somite stages. (A,B) Expression of Fgf8 at the four-somite stage. Insets in the images show expression of Hoxb1 (arrowheads) and Fgf8 (brackets) in six- to seven-somite stage embryos. Note that in the Gbx2^–/– embryo (inset in C), broad and weak Fgf8 expression forms a gradient in the anterior hindbrain with highest expression overlapping with Hoxb1 expression in r4. By contrast, Fgf8 is expressed broadly in the anterior neuroectoderm of Gbx2^–/–; Otx2^hOtx1/hOtx1 embryos (inset in D) and its posterior limit ends a few cell diameters from Hoxb1 expression in r4. (E-H) Expression of Wnt1 in the posterior hindbrain (arrows) remains unchanged in embryos that lack Otx2 (F), Gbx2 (G) or both (H) at the four- to five-somite stage. The transverse band of Wnt1 expression in the mesencephalon (brackets), however, is affected in these embryos. Inset in F shows an Otx2^hOtx1/hOtx1 embryos at the eight-somite stage with Wnt1 expression in the lateral edges of the neural plate extending from the posterior hindbrain to the anterior extreme of the embryo. (I-L) and (M-P) Expression of Pax2 at the LHF stage and four-somite stage, respectively, in embryos lacking Otx2 (J,N), Gbx2 (K,O) or both genes (L,P). The mes-met expression of Pax2 is indicated by a bracket. Expression of Pax2 in the pre-otic ectoderm is marked by arrows. (Q-T) En1 expression (brackets) is shifted anteriorly in embryos that lack Otx2 (R,T), whereas En1 expression is expanded posteriorly in embryos that lack Gbx2 (S,T). The En1 expression level in Otx2^hOtx1/hOtx1 embryos is also reduced. (U-W) Human OTX1 is expressed from the Otx2 locus in Otx2 heterozygous (U), Otx2^hOtx1/hOtx1 (V) and Gbx2^–/–; Otx2^hOtx1/hOtx1 (W) embryos. Human OTX1 is expressed only in the anteriormost endoderm and ectoderm (arrowhead), but not in the neuroectoderm of Otx2^hOtx1/hOtx1 embryos, whereas in Gbx2^–/–; Otx2^hOtx1/hOtx1 embryos human OTX1 is expressed in a broad anterior domain of the neuroectoderm. Note that all the Otx2^hOtx1/hOtx1 and Gbx2^–/–; Otx2^hOtx1/hOtx1 embryos have a similar anterior truncation (compare embryos in the second column with those in the forth column). The number of somites in the embryos is indicated in the lower right-hand corner of each panel. Anterior is towards the left, except for I-L,Q-T, which are dorsal views of embryos with anterior to the top.

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