MicroCT-Based Phenomics in the Zebrafish Skeleton Reveals Virtues of Deep Phenotyping in a Distributed Organ System

Abstract

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

The Reissner Fiber is Highly Dynamic in vivo and Controls Morphogenesis of the Spine

“…MicroCT scanning was performed as previously described [34]. All analyses were for vertebrae 1-16 [34]. Computation of standard scores, z-scores, and statistical testing using the global test were performed as previously described [34,35].…”

“…MicroCT scanning was performed as previously described [34]. All analyses were for vertebrae 1-16 [34].…”

“…All analyses were for vertebrae 1-16 [34]. Computation of standard scores, z-scores, and statistical testing using the global test were performed as previously described [34,35]. Wild type AB zebrafish were incrossed and one-cell embryos were injected with 1nL of injection mix containing 5µM EnGen Spy Cas9 NLS (NEB #M0646), 100 ng/µL sgRNA, and 25 ng/µL donor plasmid.…”

Decoding G0 somatic mutants through deep phenotyping and mosaic pattern analysis in the zebrafish skeleton

“…The past decade has seen a steady rise in genomics, fueled by advances in next-generation sequencing that have improved the speed and efficiency by which we can sequence genomes. Analogous to innovations in genome sequencing, advances in phenomics-i.e., in depth phenotyping at a large number of anatomical sites-can advance our understanding of how genetic variation influences phenotype, including biology that involves relationships between phenotypes across the whole organism (1).…”

“…Here, we identify patterns of mosaicism in the skeletons of CRISPR-edited somatic zebrafish, and relate these clonal distributions to phenotypes in mosaic models of brittle bone diseases. We employ a microCT-based workflow enabling profiling of hundreds of measures per fish (1) to characterize quantitative phenotypic variation, and perform experimentally-informed simulations to test methods for discerning and interpreting somatic mutant populations. Finally, we provide a case study for these methods by identifying and characterizing a novel zebrafish axial skeletal mutant whose target gene has been linked to genetic risk for osteoporosis.…”