WT human SIX3 is biologically potent in zebrafish embryos. (A–N) Phenotypes, classification criteria and molecular validation underlying a biosensor assay based on rescue of eye formation [rescue assay: (A–F)] and a second biosensor assay based on embryonic dorsalization [overexpression assay: (G–N)]. Embryos were injected with various combinations of an antisense MO targeting translation of the Hdl/Tcf3 protein and/or (WT) SIX3 mRNA and incubated until the mid-gastrula stage for WISH with chordin (43) and bmp2b (41) probes (K–N), at the bud stage for WISH with a wnt1 (42) probe (D–F) or until the second day of development for live scoring (A–C and G–J). Rescue assay (A–F): Hdl MO alone caused an eyeless phenotype (B) while co-injection of 50 pg WT human SIX3 mRNA efficiently restored eye development (C). WISH confirmed that injection of Hdl MO alone (E) expanded wnt1 expression levels relative to uninjected embryos in 63% (N = 30) of the embryos examined (D), although wnt1 staining intensity in both groups displayed some variability. Co-injection of SIX3 mRNA reversed this expansion and completely shut down wnt1 expression in 93% (N = 27) of the embryos examined (F). Overexpression assay (G–N): injection of 50 pg SIX3 alone produced a range of phenotypes (H–J) that we grouped into three classes. These SIX3-induced phenotypes match classical embryonic dorsalized phenotypes seen, for instance, in BMP pathway mutants (35,36). WISH on mid-gastrula-stage embryos injected with 100 pg SIX3 confirmed dorsalization on the molecular level, with clear ventral expansion of the dorsal marker chordin in 65% (N = 26) of SIX3-injected embryo (L) and 0% (N = 24) of control embryos (K). Similarly, ventral reduction of the ventral marker bmp2b was apparent in 73% (N = 26) of SIX3-injected embryos (N), compared with 10% (N = 21) of control embryos (M). Embryos in (D–F) are presented in a dorsal view, in (K and L) are presented in an animal pole view, with dorsal to the right and in (M and N) are presented in a lateral view, with dorsal to the right. White arrows in (K) and (L) point to the ventral limits of chordin stain. White arrows in (M) and (N) point to ventrally situated patches of bmp2b stain, which were lost in the majority of SIX3-injected embryos.