Whole-organism 3D Quantitative Characterization of Zebrafish Melanin by Silver Deposition Micro-CT
Melanin-rich zebrafish melanophores are used to study pigment development, human skin color, and as a large-scale screening phenotype. To facilitate more detailed whole-body, computational analyses of melanin content and morphology, we have combined X-ray microtomography (micro-CT), a non-destructive, full-volume imaging modality, with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, for both dramatic and subtle phenotypes not previously described. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, three-dimensional computational phenomic analysis of a particular cell type at cellular resolution, with potential applications in other model organisms and human melanoma biopsies. Whole-organism, high-resolution phenotyping is a challenging ideal, but provides superior context for functional studies of mutations, diseases, and environmental influences.
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In this paper, Katz and colleagues present an extension of their previous CT methods to now image melanin, a biologically important molecule in melanocytes and melanoma. This work very nicely demonstrates the way in which micro-CT can be applied to very specific biological questions in the context of a whole animal, and will be useful to those working in zebrafish, melanocyte biology and imaging fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
Reviewer #1 (Public Review):
In this paper, the authors use an innovative staining method to visualize melanin distribution for in vivo imaging of zebrafish using micro-CT. This is an extension of prior 3D imaging work using this animal model. They show this tool is able to increase resolution of key phenotypes in mutant models that impact these pigment-producing cells.
Reviewer #2 (Public Review):
In this paper, the authors extend their previous work using microCT in zebrafish to a new biological problem, which is how to detect and quantify melanin. This is an important question since melanin is involved in skin pigmentation, and affects things like mating and behavior. Moreover, melanin is also found in other structures such as the brain and kidney, although this is not examined. It would be useful to examine their already existing datasets for evidence of melanin at these more internal organs, since the role of melanin there is less well understood but micro CT could become a useful way of assessing this.