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

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.

SUBMITTER: Katz SR

PROVIDER: S-EPMC8445617 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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

Katz Spencer R SR Yakovlev Maksim A MA Vanselow Daniel J DJ Ding Yifu Y Lin Alex Y AY Parkinson Dilworth Y DY Wang Yuxin Y Canfield Victor A VA Ang Khai C KC Cheng Keith C KC

eLife 20210916

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 ...[more]

PMID: 34528510

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Project description:BackgroundA convergence of technological breakthroughs in the past decade has facilitated the development of rapid screening tools for biomarkers of toxicant exposure and effect. Platforms using the whole adult organism to evaluate the genome-wide response to toxicants are especially attractive. Recent work demonstrates the feasibility of this approach in vertebrates using the experimentally robust zebrafish model. In the present study, we evaluated gene expression changes in whole adult male zebrafish following an acute 24 hr high dose exposure to three metals with known human health risks. Male adult zebrafish were exposed to nickel chloride, cobalt chloride or sodium dichromate concentrations corresponding to their respective 96 hr LC20, LC40 and LC60. Histopathology was performed on a subset of metal-exposed zebrafish to phenotypically anchor transcriptional changes associated with each metal.ResultsComparative analysis identified subsets of differentially expressed transcripts both overlapping and unique to each metal. Application of gene ontology (GO) and transcription factor (TF) enrichment algorithms revealed a number of key biological processes perturbed by metal poisonings and the master transcriptional regulators mediating gene expression changes. Metal poisoning differentially activated biological processes associated with ribosome biogenesis, proteosomal degradation, and p53 signaling cascades, while repressing oxygen-generating pathways associated with amino acid and lipid metabolism. Despite appreciable effects on gene regulation, nickel poisoning did not induce any morphological alterations in male zebrafish organs and tissues. Histopathological effects of cobalt remained confined to the olfactory system, while chromium targeted the gills, pharynx, and intestinal mucosa. A number of enriched transcription factors mediated the observed gene response to metal poisoning, including known targets such as p53, HIF1α, and the myc oncogene, and novel regulatory factors such as XBP1, GATA6 and HNF3β.ConclusionsThis work uses an experimentally innovative approach to capture global responses to metal poisoning and provides mechanistic insights into metal toxicity.

Project description:BackgroundBone functional adaptation rationalises the inhomogeneous morphology found in bone. By means of computed tomography osteoabsorptiometry and micro-computed tomography, the mineralisation of the subchondral endplates and trabecular microstructure of vertebral bodies can be assessed to visualise the chronic loading conditions bone endures over time. In this study, we determined cancellous and compartment-specific trabecular architecture in the cervical vertebra to aid with successful integration of orthopaedic implants.MethodsWe examined the micro-computed tomography scans of seven prospectively healthy C4 vertebrae, evaluated their microstructure parameters (bone volume fraction (BV/TV), bone surface density (BS/BV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number per volume (Tb.N), connectivity density (Conn.D), structure model index (SMI), and degree of anisotropy (DA), and compared the trabecular architecture in twelve predefined volumes of interest: the cranial and caudal 0-10%, 10-15%, and 25-50% in both the ventral and dorsal half. Using computed tomography osteoabsorptiometry, the subchondral bone mineralisation of the subchondral endplates of nine C4 vertebrae was also evaluated.ResultsHighest mineralisation is located dorsally at the endplates. Tb.Sp and Tb.N were the only two parameters that displayed significant differences in averaged values of VOI. Nonetheless, distinct, consistent ventral-dorsal modulations were seen in matched sample ventral-dorsal comparison in the BV/TV, BS/BV, and SMI overall levels, as well as in Tb.Th in the three caudal levels. To simplify, the vertebra was split into ventral-cranial, dorsal-cranial, ventral-caudal, and dorsal-caudal equal quarters. The ventral quarters display lower BV/TV, respectively, higher BS/BV and SMI than their sample paired dorsal quarters. The ventral-cranial quarter shows the lowest BV/TV and the highest BS/BV and SMI, describing spacious cancellous bone with rod-like trabeculae. In contrast, the dorsal-caudal quarter exhibits the highest BV/TV and Tb.Th and the lowest BS/BV and SMI, illustrating thicker, denser, and more plate-like trabeculae. The dorsal-cranial and ventral-caudal quarters are comparable and represent intermediate characteristics.ConclusionsCT-OAM and µCT demonstrate the interdependence of compact and trabecular bone in response to long-term loading conditions. Results show highest mineralisation in the dorso-caudal part of the C4 vertebra. Recommended placement of orthopaedic implants should be positioned dorsally with screws anchored in the dorsal-caudal region.

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

Publications

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

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