Both z-Idua enzymatic activity and the accumulation of heparan sulfate (HS) in zebrafish embryos were closely correlated with knockdown or overexpression of z-idua mRNA injected into zebrafish embryos. (A) Zebrafish embryos at one-cell stage were injected with different doses of z-idua-MO, as indicated, followed by detection of z-Idua enzymatic activity at 24 hpf. The z-Idua enzymatic activity of the untreated control group was normalized as 1 for comparison with embryos injected with different doses of z-idua-MO. (B) The z-Idua enzymatic activity of zebrafish embryos injected with different doses of z-idua mRNA, as indicated. The z-Idua enzymatic activity of the untreated control group was normalized as 1 for comparison with the embryos injected with different doses of z-idua mRNA. (C) HS concentration was detected at 120 hpf of zebrafish embryos injected with 12 ng of z-idua-MO. The HS concentration of the untreated control group was normalized as 1 for comparison with the z-idua-MO-injected embryos. One trial experiment was carried out for 40 larvae. Data of each group were averaged from three independent experiments. Student’s t-test was used to determine significant differences between the two groups (**, p < 0.01).

The z-Idua enzymatic activity and occurrence of defective phenotypes in zebrafish embryos was impacted by injection of mutated z-idua mRNA into embryos. (A) The z-Idua enzymatic activity of untreated zebrafish embryos at 24 hpf, which served as the control group, was quantified and normalized as 1 for comparison with the relative z-Idua activity obtained from the other experimental groups. The z-Idua enzymatic activity of embryos injected with 100-pg zebrafish idua mRNA (z-idua; served as positive control) and various point-mutated z-idua mRNAs, as indicated, were quantified. Each point-mutation that occurred in z-idua corresponded with that of human MPS I patients. (B) Embryonic phenotypes were examined at the 120 hpf zebrafish embryos injected at one-cell stage with 100-pg zebrafish idua mRNAs (z-idua), including mutated variants, as indicated. Each mutated zebrafish idua.

Knockdown of z-idua caused defective development in zebrafish embryos. Embryonic phenotypes were observed in 120 hpf zebrafish embryos injected with 12 ng z-idua-MO. (A) Untreated control embryos; (B,C): z-idua-MO-injected embryos; (B) defective head, including smaller eyes, abnormal craniofacial and pharyngeal arches, and cardiac edema; (C) defective body shape, including shortened somites and bent body axis. (D) Percentage of defective phenotypes occurring in all examined embryos injected with z-idua-MO. Embryonic morphology was examined at 120 hpf in the zebrafish embryos injected at one-cell stage with different concentrations of z-idua-MO, as indicated. The occurrence rates (in percentages) of defective phenotypes (marked in black boxes) and the wild type-like phenotype (marked in grey) among the total examined embryos injected were calculated. The total number (n) of embryos studied in each group was indicated at the top of each column. Scale bar: 500 μm.

Knockdown of z-idua caused defective head cartilage in zebrafish embryos. Using alcian blue staining to detect the head cartilage of zebrafish embryos at 120 hpf. Untreated embryos (control) and embryos injected with antisense morpholino oligonucleotides (MO) complementary with z-idua (z-idua-MO) were studied. (A,B) Lateral view; (C,D) Ventral view; (A,C) Untreated control embryos; and (B,D) z-idua-MO injected embryos. Loss of function of z-Idua caused deformed cranial cartilage and decreased number of cartilage cells. cb, ceratobranchial; ch, ceratohyal; ep, ethmoid plate; m, Meckel’s cartilage; pq, palatoquadrate; tr, trabecular plate. Scale bar: 100 μm.

Knockdown of z-idua caused defective eyes in zebrafish embryos. Using hematoxylin and eosin staining (HE stain) and immunofluorescent staining to examine the ocular morphology of zebrafish embryos at 120 hpf. (A–D) untreated control groups; (E–H) z-idua-knockdown embryos. (A,E) HE staining; (B,F) immunofluorescent stain using zpr3 antibody to detect rod cells labeled in red fluorescence; (C,G) DAPI-marked nucleus labeled in blue fluorescence; (D,H) two signals merged. (I) To quantify the width of photoreceptor layer. (J) To quantify the cell number in retinal ganglion layer (RGL). All data were averaged from five independent experiments and represented as mean ± S.D. Student’s t-test was used to determine significant differences between each group (**, p < 0.01). ONL, outer nuclear layer; INL, inner nuclear layer; LE, lens; RGL, retinal ganglion layer; IPL, inner plexiform layer.