Fig. 3. Effects of lithium treatment carried out at a series of successive stages in Xenopus embryonic development. Stages indicate the stage at which lithium treatment was carried out (stage 6.5-10). Whole-mount RNA in situ hybridisation was carried out on all embryos at gastrulation stage 11. In all embryos dorsal is towards the top. The molecular markers used were chordin (Chd) and Xnot, which are dorsal specific, and XmyoD and Xpo, which mark ventral and lateral. Treatment with lithium at early blastula stages (A,G,M,S) results in dorsalisation of the embryo with expansion of Chd and Xnot into the ventral and lateral sides of the embryo and reduction or loss, respectively of the ventral and lateral markers Xpo and XmyoD. Treatment with lithium at early gastrula stage (F,L,R,X) results in ventralisation of the embryo with loss or reduction of the dorsal marker Xnot and expansion of the ventral and lateral markers XmyoD and Xpo into the dorsal midline. Chordin acts as a control during lithium treatment of late blastula stages as it has previously been shown that chordin is unaffected by late blastula Wnt signalling (see also Fig. 2E). Therefore the expression of chordin remains unchanged in the dorsal side of the embryo. Analysis of molecular markers shows the change of response to Wnt signalling from early dorsal-promoting to later ventrolateral-promoting is different for different Wnt responsive genes. The dorsal markers Chd and Xnot show ectopic expression in early to mid-blastula stages of development (A-C,G-I) with normal expression when lithium treated at stage 9.5 for Chd (E) and reduced expression at stage 10 for Xnot (L). Lithium treatment at stage 7.5 causes a reduction in the expression of XmyoD dorsolaterally (N). Treatment at stages 9-10 results in ventrolateral promoting response with ectopic expression in the dorsal side of the embryo (P-R). Treatment with lithium, even at the very early stages of development, results in the expansion of expression of the ventrolateral marker Xpo into the dorsal midline (T-X).