Abstract
Vertebrate models provide indispensable paradigms to study development and disease. Their analysis requires a quantitative morphometric study of the body, organs and tissues. This is often impeded by pigmentation and sample size. X-ray micro-computed tomography (micro-CT) allows high-resolution volumetric tissue analysis, largely independent of sample size and transparency to visual light. Importantly, micro-CT data are inherently quantitative. We report a complete pipeline of high-throughput 3D data acquisition and image analysis, including tissue preparation and contrast enhancement for micro-CT imaging down to cellular resolution, automated data processing and organ or tissue segmentation that is applicable to comparative 3D morphometrics of small vertebrates. Applied to medaka fish, we first create an annotated anatomical atlas of the entire body, including inner organs as a quantitative morphological description of an adult individual. This atlas serves as a reference model for comparative studies. Using isogenic medaka strains we show that comparative 3D morphometrics of individuals permits identification of quantitative strain-specific traits. Thus, our pipeline enables high resolution morphological analysis as a basis for genotype-phenotype association studies of complex genetic traits in vertebrates.
Highlights
Genotype-phenotype association is of fundamental importance for many studies, for example etiology of disease
Specimens are subjected to sample preparation, followed by tomographic scan acquisition and data processing for 3D tomographic image reconstruction and segmentation and morphometric analysis
To visualize whole adult teleosts with sufficiently high spatial resolution and highest achievable contrast, both between and within various organs, and to enable automatic segmentation, we investigated alternative staining procedures to optimize absorption contrast
Summary
Genotype-phenotype association is of fundamental importance for many studies, for example etiology of disease Such associations are facilitated by small tissue volume and transparency to visual light. Previous quantitative phenotype studies did not use whole-body morphometrics, but rather relied on landmark-based approaches with linear measurements to partition traits into different components[7]. Such approaches are limited by the resulting lack of a description of the entire anatomical phenotype in three-dimensions (3D). To ensure applicability of the methodology to comparative morphometrics with large numbers of individuals, we optimized our pipeline with respect to the chosen spatial resolution and tissue contrast in such a way that it enables application of suitable algorithms for automated segmentation of 3D data and subsequent quantitative morphometric analysis of adult teleosts
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