Fig. 3. Gain of Wnt signalling is stage specific. Various gastrula stage embryos were exposed to LiCl and the gain of Wnt activity was analysed at the nine-somite stage embryos (A-L). Untreated embryos (control) (A,G), embryos treated with LiCl at shield (B,H), 70% epiboly (C,I), 80% epiboly (D,J), 90% epiboly (E,K) and 100% epiboly stage (F,L). The expansion of midbrain and loss of eye primordia phenotypes are verified with eng2 and rx1 expressions in embryos exposed to LiCl during gastrulation (H-L) and in untreated control (G). Embryos exposed to LiCl before the mid-gastrula stage exhibit a complete loss of eye primordia (B,C,H,I), while embryos exposed to LiCl at late gastrula onwards show partial loss of eye primordia (D,E,J,K). By contrast, embryos exposed to LiCl at the end of gastrulation show almost normal anterior brain structures including eye primordia (F,L). Arrowheads indicate caudal defect in eye primordia. Scale bar: 250 µm. Anterior brain patterning is regulated by posteriorizing activity of Wnt signalling at late gastrula (M-X'). Changes of pax6 expression (blue) in the diencephalon and pax2 expression (red) in the MHB were analysed in the one-somite (M-R) and seven-somite stage embryos (S-X'), respectively. In diencephalon, pax6 expression is most abundant in the posterior diencephalon domain (arrow) and gradually decreases anteriorly. Untreated embryos (control) (M,S,S'), embryos exposed to LiCl at 60% (N,T,T'), 70% (O,U,U'), 80% (P,V,V'), 90% (Q,W,W') and 100% epiboly (R,X,X'). In embryos treated with LiCl at early gastrula, the forebrain (telencephalon, ventral diencephalon and posterior diencephalon) and eye primordia fates are lost or severely reduced, but MHB domain is expanded anteriorly (N,O,T,U,T',U'). By contrast, embryos exposed to LiCl at 80% (P,V,V') and 90% epiboly (Q,W,W'), the posterior diencephalon fate is expanded without increasing MHB domain. Embryos exposed to LiCl at the 100% epiboly show almost normal anterior brain patterning at the seven-somite stage (X,X'). Scale bar: 125 µm.

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