Utility of quantitative micro-computed tomographic analysis in zebrafish to define gene function during skeletogenesis

Abstract

The zebrafish is a powerful experimental model to investigate the genetic and morphologic basis of vertebrate development. Analysis of skeletogenesis in this fish is challenging as a result of the small size of the developing and adult zebrafish. Many of the bones of small fishes such as the zerbafish and medaka are quite thin, precluding many standard assays of bone quality and morphometrics commonly used on bones of larger animals. Microcomputed tomography (microCT) is a common imaging technique used for detailed analysis of the skeleton of the zebrafish and determination of mutant phenotypes. However, the utility of this modality for analysis of the zebrafish skeleton, and the effect of inherent variation among individual zebrafish, including variables such as sex, age and strain, is not well understood. Given the increased use and accessibility of microCT, we set out to define the sensitivity of microCT methods in developing and adult zebrafish. We assessed skeletal shape and density measures in the developing vertebrae and parasphenoid of the skull base. We found most skeletal variables are tightly correlated to standard length, but that at later growth stages (>3 months) there are age dependent effects on some skeletal measures. Further we find modest strain but not sex differences in skeletal measures. These data suggest that the appropriate control for assessing mutant phenotypes should be age and strain matched, ideally a wild-type sibling. By analyzing two mutants exhibiting skeletal dysplasia, we show that microCT imaging can be a sensitive method to quantify distinct skeletal parameters of adults. Finally, as developing zebrafish skeletons remain difficult to resolve by radiographic means, we define a contrast agent specific for bone that enhances resolution at early stages, permitting detailed morphometric analysis of the forming skeleton. This increased capability for detection extends the use of this imaging modality to leverage the zebrafish model to understand the development causes of skeletal dysplasias.

Zebrafish: An Emerging Model for Orthopedic Research

“…The three‐dimensional (3D)‐high‐resolution micro‐computed tomography has become established as a powerful method to assess bone morphology and microstructure in zebrafish . Using a 5 μm voxel size, bone structure indices as vertebral bone volume, thickness, and eccentricity can be characterized .…”

“…Using a 5 μm voxel size, bone structure indices as vertebral bone volume, thickness, and eccentricity can be characterized . Neural arch area, which reflects modeling arising from osteoblast and osteoclast activity, can also be captured . Because of their small size, whole body, high‐resolution scans are readily acquired .…”

Integration of multiple imaging platforms to uncover cardiac defects in adult zebrafish

“…We complemented this functional analysis with ex-vivo µ-CT imaging to examine the morphology of the adult zebrafish heart in fine detail. With a few exceptions (Descamps et al, 2014;Vagberg et al, 2015;Babaei et al, 2016;Weinhardt et al, 2018;Ding et al, 2019), this technique has been mainly used for ex-vivo samples focusing on the skeleton (Charles et al, 2017;Hur et al, 2017;Silvent et al, 2017;C. J. Watson et al, 2020).…”

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

“…BMP1 is a secreted enzyme that functions in the cleavage of C-propeptides from procollagen precursors (28). We and others previously showed that zebrafish germline loss-of-function mutants for plod2 and bmp1a exhibit severe skeletal abnormalities as adults, reminiscent of OI phenotypes (1,27,29,30).…”