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.

Comparison of the two biosensor assays. Various doses of WT SIX3 mRNA were injected into embryos either with (for rescue assay calibration) or without (for overexpression assay calibration) co-injection of 1 ng Hdl MO. The Y-axis represents the fraction of embryos that achieved the theoretical maximum score on two distinct criteria scales. For the rescue assay, the measurable parameter was the presence of rescued eyes with the theoretical maximum of 100%. For the overexpression assay, the measurable parameter was the distribution of phenotypic classes that were seen and the theoretical maximum was a PI score (see Materials and Methods) of 3, which would be achieved if exclusively class 3 embryos were obtained. Note the rapid transition from half of the embryos rescued by 2.5 pg SIX3 mRNA to nearly all rescued by 5.0 pg SIX3 mRNA, explaining the high variability we observed with this assay (Fig. 5).

(A–C) Relative strengths of the 46 variant SIX3 alleles tested (see Table 1 for clinical details). Open circles represent the individual experiments. The Y-axis represents normalized outcomes, as explained in Material and Methods. Filled circles represent the average outcome and the vertical bars represent the 95% confidence interval, calculated by simulation using a multinomial model. Raw values are reported in Supplementary Material, Table S1. Alleles are presented 5′–3′. (A) Alleles 1–16 (see Table 1 and Supplementary Material, Table S1 for clinical details), spanning the N-terminus, the poly-glycine domain and N-terminal half of the Six domain. (B) Alleles 17–32, spanning the C-terminal half of the SIX domain and N-terminal half of the homeodomain. (C) Alleles 33–46, spanning the C-terminal half of the homeodomain, the C-terminal motifs and the C-terminus. Data points are color-coded to represent allele strengths in the overexpression assay, as also indicated in Table 1 and Fig. 4: color: red = <50% activity, orange = 50–90% activity and black >90% activity.

Summary of structural alterations detected in the human SIX3 protein. A virtual translation of the human cDNA sequence used for our functional analysis is depicted with positions of variations according to individual mutation number (Table 1) and color representing activity in the overexpression assay: red = <50% activity, orange = 50–90% activity and black = >90% activity. An N-terminal poly-glycine segment (gray) is the most striking difference between multiple-species alignments and is present in human and rodent sequences but is largely absent in chick, zebrafish and Xenopus (data not shown). An uncommon length variant 205–207dupGGC adds an additional Glycine69 residue and was found in normal controls and can explain minor numbering differences among different SIX3 mutation reports. The canonical SIX domain (blue and underlined) also contains two eh1-like motifs (salmon-colored); note functional mutations were detected in both motifs. The homeodomain (purple and italic) is a target for in-frame deletions (e.g. 34**) and frameshift (e.g. 25*), whereas typical premature termination signals are indicated by the mutation number and the hash symbol. Note two putative C-terminal motifs (green) (38) are also functionally implicated by mutations leading to impaired activity.

Side-by-side comparison of relative signaling strengths for various predicted SIX3 truncation alleles in the two biosensor assays. Open circles represent individual experiments. The Y-axis represents the normalized outcomes, as explained in Material and Methods and Supplementary Material, Table S2. Filled circles represent the average outcome and the vertical bars represent the 95% confidence interval, calculated by simulation using a multinomial model. The two most N-terminal truncations (patients 8 and 11), as well as point mutations in the first eh1-domain (patients 4 and 5), cause loss of function in both assays; however, downstream truncations in which the N-terminal 129 amino acids are intact generally retained activity in the rescue assay (patients 12, 15, 16, 19, 23 and 24, but not 17), as did a point mutation in the second eh1 domain (patient 18).