Fig. 4. Expression of placodal and hindbrain marker genes in double mutant embryos at E8.0 reveals a disturbance of placodal patterning in the absence of hindbrain patterning defects. Whole mount embryos were probed with labelled cDNA for Pax2 (A,C), Pax8 (E,G), Dlx5 (I,K), Gbx2 (M,O), HoxB1 (Q,R), kr (S,T) and Krox20 (U,V). Embryos probed with placodal markers were sectioned in the transverse plane. A section taken through the otic region (the plane is indicated by a line through each embryo) is shown in the panel to the right of each whole embryo. Rostral is to the left. In situ hybridisation with Pax2 to 8 somite control (A) and double mutant (C) embryos detects transcripts in the eye (e), kidney (k) and isthmus (i). Pax2 transcripts can be detected in the otic placode (op) in control (B) but not double mutant (D) embryos. At 8 somites, Pax8 transcripts can be detected in control embryos in the otic placode and more ventrally in the surface ectoderm (F). In double mutant embryos, Pax8 transcripts can only be detected in the more ventrally located surface ectoderm (se, H). In situ hybridisation with Dlx5 to 10 somite control (I) and double mutant (K) embryos detects transcripts in the forebrain (fb). Dlx5 transcripts can be detected in control embryos in the otic cup (oc) (J), but in double mutant embryos the region of Dlx5 expressing thickened ectoderm is located more ventrally (L). At 6 somites, Gbx2 is expressed throughout the surface ectoderm including the surface ectoderm and in the underlying mesoderm (m) (N). In double mutant embryos, Gbx2 transcripts are excluded from the most dorsal regions of the surface ectoderm and from the underlying mesoderm of the otic region (P). At E9.0 HoxB1, Mafb/kreisler and Krox20 are expressed in rhombomeres 4 (r4; Q), rhombomeres 5/6 (r5/6; S) and rhombomeres 3 and 5 (r3, r5; U), respectively. Expression of HoxB1, Mafb/kreisler and Krox20 is unchanged in double mutant embryos (R,T,V). A bracket marks the location of the dorsal surface ectoderm of double mutant embryos, from which otic marker genes are excluded.

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