Fig. 3

Fig. 3

GNAQ^Q209P rescues the hypopigmentation phenotype of golden (slc24a5^-/-) zebrafish. A.I, B.I, C.I. Lateral views of wild-type, golden, and Tg (mitfa:GNAQ^Q209P;slc24a5^-/-) 5 dpf zebrafish embryos, respectively. A.II, B.II, C.II. Top views of wild-type, golden, and Tg (mitfa:GNAQ^Q209P;slc24a5^-/-) 5 dpf zebrafish embryos, respectively. Scale bars, 100 μm. D.I, E.I, F.I, G.I. Lateral views of 2-month-old adult zebrafish. (D.I) Wild-type illustrating normal melanin pigmentation in skin melanocytes. (E.I) Light-skinned golden mutant (slc24a5^-/-) with stripes comprising less melanin-rich melanocytes. (F.I, G.I) Two examples of Tg (mitfa:GNAQ^Q209P;slc24a5^-/-) zebrafish from independent founders, acquiring darker stripes as compared to wild-type (D.I) and golden mutants (E.I), but still conserving the normal striped pigment pattern. Scale bars, 0.5 cm. D.II, E.II, F.II, G.II. H&E staining of transverse sections of formalin-fixed and paraffin-embedded eye specimens of 2-month-old adult zebrafish. (D.II) Normally pigmented RPE located between the photoreceptors layer of the retina (rods & cones) and the choroid in a wild-type zebrafish. (E.II) Lightly pigmented RPE in a golden mutant. (F.II, G.II) Rescuing of hypopigmented RPE in both GNAQ^Q209P-expressing golden mutants, with no pathological findings yet observed in the choroidal melanocyte layer. Scale bars, 50 μm. H.I, I.I, J.I, K.I. Tail fins of wild-type, golden, and two Tg (mitfa:GNAQ^Q209P;slc24a5^-/-) zebrafish, respectively. These were dissected, fixed, and sectioned for TEM analysis. Black dashed boxes indicate representative regions of the tail fins examined by TEM. Scale bars, 0.5 cm. H.II, I.II, J.II, K.II. Transmission electron micrographs illustrating the structure of melanosomes in the melanocytes of dissected tail fins. Melanosomes in golden mutants (I.II) are less densely pigmented as compared to those of wild-type (H.II) and transgenic zebrafish (J.II, K.II). Scale bars, 200 nm. L. Relative to zebrafish EF1alpha (EF1α) expression and as compared to non-injected golden controls, qRT-PCR data show significant increase in mRNA expression levels of GNAQ in Tg (mitfa:GNAQ^Q209P;slc24a5^-/-) zebrafish. In contrast, no significant alterations were observed in the expression of mitfa or downstream differentiation genes including tyrosinase, tyrp-1, and dct. Results represent mean ± SEM of triplicates of three independent experiments. ***P <0.05, using two-tailed, unpaired t test. Abbreviations: tyrp-1, tyrosinase-related protein 1; dct, dopachrome tautomerase.