HPE-Associated Six3 Mutants Function as Hypomorphs

(A–D) The five generated six3b HPE-related mutant forms can rescue the headless phenotype in injected hdl/tcf3^-/- mutant embryos. ddH₂O and mGFP (250 pg) were injected as negative controls. Rescue was scored at 55 hr postfertilization (hpf) according to three phenotypic categories: no rescue (yellow) (A), partial rescue (light blue) (B), and full rescue (dark blue) (C). The results are graphically represented in (D), which shows the samples and doses injected.

(E–H) The six3b mutant forms show hypomorphic activity in suppressing the eyeless phenotype of six3b;six7 mutant morphants. The six3b^vu87/vu87 mutants injected with six7 antisense morpholino oligonucleotide (MO) exhibit an eyeless phenotype. After coinjection of six3b mutants with six7 MO and six3b wild-type or mutant forms, embryos were graded according to the size of their eyes (0–6.5 point [pt] scale). Examples are shown of absent eyes (0 pt) (E), severely reduced eyes (arrowhead) (1.2 pt) (F), moderately reduced eyes (3.0 pt) (G), and full-size eyes (5.3 pt) (H).

(I) Graphical interpretation of these results. The E109Stop null mutation found in six3b^vu87 mutants was overexpressed as a negative control. Double asterisks indicate eye size significantly different from that of six3b^vu87/vu87-six7 mutant morphants (p ≤ 0.01). The absence of asterisks on mutant form-injected samples indicates a p value that exceeds 0.05 (statistically insignificant).

(J) Summary of the three zebrafish-based functional assays. Five HPE-associated Six3 mutants can be separated into three groups on the basis of their hypomorphic activity.

HPE-Associated Six3 Mutants Function as Hypomorphs
(A) Schematic representation of wild-type and HPE-associated SIX3-mutant proteins. The black box represents the Six domain. The three alpha helixes of the homeodomain are indicated.
(B-F) The injected Six3-mutant proteins showed a spectrum of hypomorphic activities in the zebrafish assay that ranged from a slightly decreased ability to dorsalize embryos (Group 1: Six3bV92G), to a greatly decreased dorsalizing potential (Group 2: Six3bV250A, Six3bH173P), to an inability to dorsalize embryos at the injected doses (Group 3: Six3bR257P, Six3bGGins). Wild-type six3b (B) and Group 1 (C) and Group 2 (D) six3b-mutant forms can morphologically dorsalize wild-type embryos into a characteristic egg-like shape, as seen at the 1-somite/10.3-hpf stage. (E) Group 3 mutant forms fail to dorsalize embryos even at very high injection doses (240 pg-1 ng). (G, H) The expression of chd (dorsal marker) and wnt8a (ventral marker) is shown by wholemount in situ hybridization at the 60% epiboly/6.5-hpf stage. (I, J) Wild-type six3b injection shows the expansion of chd around the margin of the embryo, and the downregulation of wnt8a expression. (K-N) Misexpression of Groups 1 and 2 mutant forms causes chd expansion and wnt8a loss similar to wild-type six3b, although higher concentrations were needed. (O, P) Embryos misexpressing group 3 mutant forms have chd and wnt8a expression patterns similar to those seen in uninjected embryos. (Q) Graphical representation of results obtained following injections of equivalent amounts (240-300 pg) of six3b-mutant forms and wild-type six3b. Wild-type six3b and injection of Groups 1 and 2 mutant forms show a statistically significant difference (p ≤0.01) compared to uninjected wild-type embryos; however, injection of Group 3 did not (p >0.05).