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

Spencer R Katz,Dilworth Y Parkinson,Yuxin Wang,Victor A Canfield,Maksim A Yakovlev,Daniel J Vanselow,Yifu Ding,Keith C Cheng,Alex Y Lin,Khai C Ang

doi:10.7554/elife.68920

Spencer R Katz, Dilworth Y Parkinson + Show 8 more

Open Access

https://doi.org/10.7554/elife.68920

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Abstract

We previously described X-ray histotomography, a high-resolution, non-destructive form of X-ray microtomography (micro-CT) imaging customized for three-dimensional (3D), digital histology, allowing quantitative, volumetric tissue and organismal phenotyping (Ding et al., 2019). Here, we have combined micro-CT with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. The resulting images enabled whole-body, computational analyses of regional melanin content and morphology. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and further allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, including subtle phenotypes not previously noticed. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, 3D computational phenomic analysis of a specific cell type at cellular resolution, with potential applications in other model organisms and melanocytic neoplasms. Advances such as this in whole-organism, high-resolution phenotyping provide superior context for studying the phenotypic effects of genetic, disease, and environmental variables.

Highlights

The zebrafish (Danio rerio) is an important vertebrate model organism for developmental biology, functional genomics, and large-scale drug and toxicological screens due to its high fecundity, transparent, externally developing embryos, relatively low husbandry costs, and small size (Lieschke and Currie, 2007)
Vertebrate models such as zebrafish play a key role in understanding human health and disease by elucidating relationships between mutations or environmental exposures and observable phenotypic changes, including pigmentation-related disorders such as albinism and melanoma
We investigated whole-animal melanin pigmentation in wild-type and mutant zebrafish through the novel application of ionic silver staining for micro-CT imaging

Summary

Introduction

The zebrafish (Danio rerio) is an important vertebrate model organism for developmental biology, functional genomics, and large-scale drug and toxicological screens due to its high fecundity, transparent, externally developing embryos, relatively low husbandry costs, and small size (Lieschke and Currie, 2007). Cell Biology | Developmental Biology mpv17a9/a9; slc45a2b4/b4) strain, a compound mutant with defects in both melanogenesis and iridophore development, never develops pigment and is used for applications where transparent post-e mbryonic organisms are required, such as fluorescence imaging (Antinucci and Hindges, 2016) Another pigmentation mutant, golden (slc24a5b1/b1), is frequently used for mutagenesis screening or testing of gene editing technology because its single affected locus produces an early, dramatic reduction in eye and body melanin content that has been implicated in human skin color (Doyon et al, 2008; Ginger et al, 2008; Hoshijima et al, 2016; Lamason et al, 2005; Moore et al, 2006; Streisinger et al, 1981; Streisinger et al, 1989). To demonstrate the utility of this technique, we imaged wild-type zebrafish as well as mutant strains with unique pigmentation patterns, including golden and body pigment-deficient mutants (nacre/casper) (D’Agati et al, 2017; Lister et al, 1999; White et al, 2008), for visualization and quantitative analyses of zebrafish melanin

Materials and methods

Results and discussion

D Light Microscopy

Conclusions and future work

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