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Fig. 2 zic gene expression is patterned in the neurectoderm during gastrulation. Staged embryos were stained for opl/zic1, zic2, zic3 (purple), and otx2 RNA (orange in G-I) by using whole-mount in situ hybridization. A-C: Early gastrula (shield) stage embryos, dorsal view, anterior at the top. A: opl/zic1 is not expressed. B: zic2 is expressed in the blastoderm margin, the presumptive mesendoderm, only. C: zic3 expression in the posterior portion of the dorsal ectoderm and in the blastoderm margin. D-F: Mid-gastrula (80% epiboly) stage embryos, dorsal view, anterior at the top. D: opl/zic1 expression restricted to an anterior domain of the neural plate. E: zic2 expression domain includes the anterior opl domain and extends more posteriorly. F: zic3 is expressed in the posterior portion of the neural plate and at the edge of the neural plate anteriorly. G-I: Late gastrula (90% epiboly) stage embryos, dorsal view, anterior at the top. G,H: Expression of opl (G) and zic2 (H), shown in purple, overlap the otx2 (orange) expression domain in the presumptive forebrain. I: anterior portion of zic3 expression overlaps the posterior portion of the otx2 domain. Dots indicate outline of the blastoderm margin. White arrowheads point to the posterior extent of otx2 staining, which corresponds to the future posterior midbrain.