Effect of human coding polymorphisms on zebrafish mRNA rescue of the albino phenotype.

Lateral views of 48-hpf (A) wild-type zebrafish larva (B) un-injected alb^nk1 zebrafish larva (C-F) alb^nk1 zebrafish larva injected with mRNA (500 pg), coding for indicated variants of zebrafish slc45a2, named according to positions of human variation (human 272 equivalent to zebrafish 303; human 374 equivalent to zebrafish 403). (C) wild-type; (D) E272K mutant; (E) L374F mutant; (F) E272K/L374F double mutant. Note that mRNA rescue in zebrafish does not occur in every cell, which is thought to be due to unequal distribution of the mRNA in the cytoplasm of the originally injected eggs, resulting in unequal distribution of the mRNA among different cells of the embryo. The results shown are typical of a majority of injected embryos in each case; each of embryo in the majority populations contains cells as pigmented as the ones evident in this figure. Scale bar 400, μm.

Effect of a human coding polymorphism on zebrafish mRNA rescue of the golden phenotype.

Lateral views of 48-hpf (A and B) wt zebrafish larva (C and D) gol^b1 zebrafish larva (E and F) gol^b1 larva injected with full-length zebrafish slc24a5 (wt) mRNA (500 pg) and (G and H) gol^b1 larva injected with full-length zebrafish slc24a5 mRNA with a single nucleotide change (500 pg), coding for the orthologous human derived A111T allele. Scale bars in (A, C, E, G) 150 μm, (B, D, F, H) 400 μm.

Injection of human mRNAs fail to rescue albino. (A) Wild-type zebrafish slc45a2 mRNA (1400 pg) injected into albino embryos rescues pigmentation (arrows) while SLC45A2 mRNA of human (B) ancestral (L374) (1400 pg) and (C) derived (L374F) (1400 pg) alleles do not. Scale bar 300 μm.

Morpholino knockdown of slc45a2 phenocopies albino. Lateral views of wild-type 48-hpf zebrafish larvae that are uninjected (A) or injected (B) with 8 ng morpholino targeted to the 5′UTR of slc45a2. Scale bar 200 μm.

Co-injection of morpholino and mRNA for slc45a2 into the zebrafish embryos causes substantial developmental defects. (A) zebrafish wild-type embryos (B) Injection of zebrafish embryos with 5′UTR morpholino (8 ng) against slc45a2 (non-overlapping with the mRNA sequence) reduces pigmentation, while (C) coinjection with slc45a2 mRNA (500 pg) causes severe developmental defects that interfere with detection of phenotypic rescue. Scale bar 600 μm.

Co-injection of morpholino and mRNA for slc24a5 into the zebrafish embryos causes substantial developmental defects. (A) zebrafish wt embryos (B) Injection of zebrafish embryos with 5′UTR morpholino against slc24a5 (non-overlapping with the mRNA sequence) reduces pigmentation and, (C) and, with coinjection with slc24a5 mRNA (500 pg) causes severe developmental defects similar to those seen in Figure S3, panel C, precluding the detection of rescue. Scale bar 300 μm.