Expression of vcanb and chsy1 in the ext2 mutant ear. (A–H)In situ hybridisation to vcanb(A–F) and chsy1(G,H) mRNA in phenotypically wild-type sibling and ext2 mutant embryos. (A) Expression of vcanb is present in the ear (black arrowhead) and pectoral fin (black arrow) in sibling embryos. (B) Only a rudimentary pectoral fin bud is present in the ext2 mutant, and the associated vcanb staining is missing (white arrow). (C,D) At 72 hpf, vcanb expression is strongly reduced in the periderm of the second arch (future operculum) in ext2 mutants [black arrowhead in (C), white arrowhead in (D)]. (C’,D’) Enlargements of the ears shown in (C,D), respectively. Expression of vcanb is now down-regulated in the pillars of the wild-type ear, remaining only in the dorsolateral septum (dls). In the ext2 mutant, expression persists abnormally in the unfused anterior and posterior projections [ap, pp, (D’)] and in an abnormal pillar-like structure (boxed insert of same ear taken at a different focal plane). (E,F)vcanb expression persists abnormally in the unfused projections in mutant ears until at least 120 hpf. (G,H) Overall levels of chsy1 expression are slightly reduced in ext2 mutants (H); expression is missing in the viscera (white arrowhead), but persists in unfused epithelial projections in the ear (black arrowhead). All panels are lateral views with anterior to the left, dorsal to the top (orientation shown in (B): A, anterior; D, dorsal). Scale bars: in (A), 100 μm (also applies to (B)); in (C), 100 μm [also applies to (D,G,H)]; in (C), 50 μm [also applies to (D’)].
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