Figure 4. Silver staining and X-ray microtomography (micro-CT) of mutant zebrafish enables comparison of melanin content and organization with wild-type larvae.

Volume renderings (A, D, G), segmented volumes (B, E, H), and representative single slices of micro-CT reconstructions through the retinal pigment epithelium (RPE; C, F, I) of representative 5 days post-fertilization (dpf) wild-type (A–C), slc24a5^b1/b1 (golden; D–E), and mitfa^w2/w2 (nacre or casper; G–I) larvae. In the segmented volumes, red = dorsal stripe, yellow = ventral stripe, green = yolk sac stripe, cyan = lateral stripes, white = RPE, gray = other body melanin, purple = lens. As compared to wild-type larvae, slc24a5^b1/b1 mutant larvae exhibit a reduction in staining throughout the body and eyes (D) but retain the overall organization of pigment layers (E). The RPE in slc24a5^b1/b1 larvae (F) is thinner than the wild-type RPE (C) with less intense staining. The mitfa^w2/w2 mutant lacks all body pigment; staining is observed only in the RPE and some argentaffin material posterior to the eyes (G–H, arrow). The RPE of the mitfa^w2/w2 mutant (I) is of similar thickness and staining intensity as the wild-type RPE (C). For each comparison, visualization settings were kept constant for the wild-type and mutant fish. § = head segment only shown. All stained samples analyzed in this study are shown in Figure 4—figure supplement 1.

Figure 4—figure supplement 1. Overview of all analyzed samples.

Light micrographs (before silver staining), X-ray microtomography (micro-CT) volume renderings, and segmented volumes of the wild-type, slc24a5^b1/b1, and mitfa^w2/w2 5 days post-fertilization (dpf) larvae (n=3 each) analyzed for this study. In the segmented volumes, red = dorsal stripe, yellow = ventral stripe, green = yolk sac stripe, cyan = lateral stripes, white = RPE, gray = other body melanin, purple = lens. Scale bars = 500 µm. § = head segment only analyzed by micro-CT and shown.