FIGURE SUMMARY
Title

Whole-organism 3D quantitative characterization of zebrafish melanin by silver deposition micro-CT

Authors
Katz, S.R., Yakovlev, M.A., Vanselow, D.J., Ding, Y., Lin, A.Y., Parkinson, D.Y., Wang, Y., Canfield, V.A., Ang, K.C., Cheng, K.C.
Source
Full text @ Elife

Ionic silver stains reproduce three-dimensional (3D) pigmentation patterns in X-ray microtomography (micro-CT) images.

(A) Schematic overview of staining procedure. Ammoniacal silver solution reacts with endogenous melanin to deposit solid silver, which attenuates X-rays in micro-CT imaging. (B) A representative cropped and rotated slice of a micro-CT reconstruction of a 5 days post-fertilization (dpf) wild-type zebrafish stained with silver. RPE = retinal pigment epithelium, P = body pigment, L = lens, r = resin, it = inner wall of sample tube, ot = outer wall of sample tube, b = air bubble. (C) 3D rendering of a 5 dpf wild-type zebrafish stained with silver with a heatmap to illustrate pigment density throughout the fish. (D) Corresponding light microscopy (left) and micro-CT (right) images exhibit the same pigmentation patterns. In the micro-CT image, a top-down 3D rendering of stained melanin is shown in grayscale with the dorsal-most melanin digitally colored red to aid comparison with the light micrograph. Some distinguishing shared features are highlighted with colored arrowheads. White arrows indicate deeper melanin obscured by soft tissue in the light micrograph that can be visualized by targeted 3D re-rendering. Scale bars = 200 µm. Unstained samples do not exhibit melanin-related attenuation (Figure 1—figure supplement 1).

Silver-based X-ray microtomography (micro-CT) enables segmentation and visualization of melanin microanatomy in three dimensions (3D).

(A) A light micrograph of the side of a 5 days post-fertilization (dpf) wild-type larva indicating the major regions of melanin pigment: dorsal, lateral, ventral, and yolk sac stripes and the retinal pigment epithelium (RPE). (B) A 3D rendering with orthoslice (C) of micro-CT volumes segmented into anatomical regions shows the organization of larval pigment into layers. Red = dorsal stripe, yellow = ventral stripe, green = yolk sac stripe, cyan = lateral stripes, white = RPE, gray = other body melanin, purple = lens. (C) Single slice of micro-CT data with color overlay corresponding to the indicated region in B. (D) 200-slice maximum intensity projection of dorsal stripe melanin exhibits transparencies in the staining indicating the position of large organelles (arrows) including potentially binucleated cells (circles). A sample of these transparencies was measured to estimate average size (Figure 2—figure supplement 1, Figure 2—source data 1). (E) View from the top-down of a volume rendering showing rostral melanin in the nose forming globular, dendritic cells (arrowheads). RPE-L = left RPE, RPE-R = right RPE. (F) Isolated volume rendering of the right eye with a clipping plane showing the villous inner surface and smooth outer surface of the RPE. The rendering has been falsely colored by intensity to highlight certain anatomical features, including local pigment thickness variability throughout the RPE, the egress of the optic nerve (arrowhead), and the fused choroidal fissure (arrows). L = lens.

Quantification of silver-stained, X-ray microtomography (micro-CT)-scanned, and segmented larvae reveals wild-type melanin volume and content trends.

Wild-type (wt) 5 days post-fertilization (dpf) larvae (n = 3) were stained with silver and micro-CT imaged under the same conditions then segmented into major pigment regions as described in Figure 2: retinal pigment epithelium (RPE, right and left), dorsal stripe (DS), ventral stripe (VS), yolk sac stripe (YSS), lateral stripes (LS, right and left), and other melanin (other). (A) Volumes of the total pigmented regions and each segmented region are shown for the three fish. (B) Reconstructed stain intensity values are assumed to be proportional to melanin density; integrated intensity values for the segmented regions represents the melanin content of these regions. As proportions of total melanin content (summed across all segmented regions) the wt fish show high concordance between individual samples. Error bars = standard deviation from average.

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), slc24a5b1/b1 (golden; D–E), and mitfaw2/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, slc24a5b1/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 slc24a5b1/b1 larvae (F) is thinner than the wild-type RPE (C) with less intense staining. The mitfaw2/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 mitfaw2/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.

Silver-based X-ray microtomography (micro-CT) enables quantitative comparisons of wild-type and mutant pigmented samples.

Wild-type (wt, n = 3), slc24a5b1/b1 (n = 3), and mitfaw2/w2 (n = 3) 5 days post-fertilization (dpf) larvae were stained with silver and micro-CT imaged under the same conditions. Normalized reconstructed intensity values (norm. values) are assumed proportional to melanin density; integrated intensity values for the segmented regions represent the melanin content of these regions. Volume and melanin content from total pigmented regions (A–B), combined right and left retinal pigment epithelia (RPE) (C–D), and pigmented regions outside the eye (E–F) are shown for all samples with percent change of mean from wt indicated. p-Values were determined by Tukey post hoc test following one-way ANOVA and considered significant at p < 0.05 (shown in red). § = head segments only analyzed. Error bars = standard deviation from average.

Acknowledgments
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