Fig. S1

The effects of heatshock-induced bmp2b and dkk1 overexpression, and SU5402 treatment on sox2, sox3 and krox20 expression and embryo phenotype. (A-F) Embryos carrying the hsp70l:bmp2b transgene were heatshocked at shield stage and fixed at 12hpf. bmp2b overexpression led to a decrease in the expression of sox2 (100%, n=8) (D versus A), sox3 (80%, n=10) (E versus B) and krox20 (100%, n=10) (F versus C). (G-L) Embryos carrying the hsp70l:dkk1 transgene were heatshocked at shield stage and fixed at 12 hpf. dkk1 overexpression led to a slight decrease in sox2 expression and a shortening of its posterior expression domain (54%, n=13) (J versus G), a slight decrease intensity of the anterior aspect of sox3 expression with little change in intensity in the posterior along with a shortening of its posterior expression domain (100%, n=12) (K versus H) and a decrease in the extent of krox20 expression (100%, n=17) (L versus I). (M-R) Embryos were treated with 90 μM SU5402 starting at 7 hpf and fixed at 12 hpf. SU5402 treatment led to a decrease in the extent and intensity of sox2 expression (P versus M) (100%, n=14) and an almost complete loss of krox20 expression (R versus O) (100%, n=16). SU5402 treatment caused an increase in the level of sox3 mRNA in the anterior neural plate, but the expression pattern was less organized (Q versus N, insets) (100%, n=16). (S-Z) The 18 hpf phenotypes of embryos undergoing various treatments are shown. The phenotype of embryos treated with SU5402 is similar to the fgf8a mutant phenotype (Brand et al., 1996), including a bulge in the midbrain primordium and defects in mesoderm development (T versus S). Embryos overexpressing bmp2b exhibit strong ventralization (U versus S). Embryos overexpressing dkk1 or injected with wnt8a MO exhibit anterior-posterior patterning defects consistent with a loss of Wnt signaling (V-Z versus S). We split embryos injected with wnt8a MO or overexpressing dkk1 into three classes of increasingly severe Wnt phenotypes (V-Z). (AA) The proportion of embryos from each treatment in each class is shown. In general, embryos injected with wnt8a MO had more severe phenotypes then those overexpressing dkk1 after a shieldstage heatshock.