Figure 2. A ZLI-like signalling centre in S. kowalevskii.

a-u, in situ hybridizations for S. kowalevskii and mouse homologs of ZLI and diencephalon markers. Arrowheads mark the proboscis/collar boundary in S. kowalevskii. Mouse images show hemisected heads at E10.5, and dashed lines indicate the ZLI with arrows denoting its extent. a, S. kowalevskii hh expression. b, S. kowalevskii ptch expression (also see Supplementary fig. 2). c, mouse shh expression. d, diagram of shh expression showing ZLI in dark blue. e, mouse ptch1 expression. f, mouse ptch2 expression. g, S. kowalevskii fng expression. h, S. kowalevskii otx expression. i, mouse otx1 expression. j, mouse otx2 expression. k, hh (magenta) and otx (green) are co-expressed at the presumptive proboscis/collar boundary. l, S. kowalevskii wnt8 expression. m, mouse wnt8b expression. n, S. kowalevskii pax6 expression. o, double in situ hybridization showing pax6 expression (fluorescence, green) anterior to hh (colorometric, black). p, mouse pax6 expression. q, S. kowalevskii dlx expression. r, double FISH showing dlx expression (green) in the proboscis base anterior to hh (magenta). s, mouse dlx2 expression. t, S. kowalevskii foxa expression. u, double FISH showing foxa (green) and hh (magenta) expression. v, diagram of AP expression topologies of ZLI and forebrain marker homologs in S. kowalevskii and mice. Six3 expression based on previous data⁷. Anterior to top. w, x, otx siRNA downregulates hh expression at the proboscis/collar boundary (w) relative to a control siRNA (x). y, z, hh siRNA reduces dlx expression in the proboscis base (y) relative to a scrambled siRNA (z). ai, bi, cyclopamine treatment reduces dlx expression in the proboscis base (ai) relative to a control embryo treated with DMSO (bi). Insets show ventral views. Scale bars = 100 μm in S. kowalevskii embryos and 1 mm in mice. Abbreviations: mge, medial ganglionic eminence; pth, prethalamus; th, thalamus; others as described previously.