Abstract
Although bone is one of the most studied living materials, many questions about the manner in which bones form remain unresolved, including fine details of the skeletal structure during development. In this study, we monitored skeleton development of zebrafish larvae, using calcein fluorescence, high-resolution micro-CT 3D images and FIB-SEM in the block surface serial imaging mode. We compared calcein staining of the skeletons of the wild type and nacre mutants, which are transparent zebrafish, with micro-CT for the first 30 days post fertilization embryos, and identified significant differences. We quantified the bone volumes and mineral contents of bones, including otoliths, during development, and showed that such developmental differences, including otolith development, could be helpful in identifying phenotypes. In addition, high-resolution imaging revealed the presence of mineralized aggregates in the notochord, before the formation of the first bone in the axial skeleton. These structures might play a role in the storage of the mineral. Our results highlight the potential of these high-resolution 3D approaches to characterize the zebrafish skeleton, which in turn could prove invaluable information for better understanding the development and the characterization of skeletal phenotypes.
Highlights
The zebrafish, a species belonging to the Cyprinidae family, is a well-studied vertebrate developmental model because of its basal phylogenetic location, because of the high degree of homology between human and zebrafish genes [1] and organ systems [2] and because of the optical clarity of its embryos and larvae, allowing in vivo observations during development [3,4,5]
We focused the comparison only on the head and the first precaudal vertebrae at 17 dpf when the head and axial skeleton are fully developed based on calcein imaging, and compared these results to 30 dpf (Fig 1)
We show here that high resolution 3D imaging using confocal microscopy, quantitative FIBSEM in the block surface serial imaging mode and quantitative high resolution laboratory based micro-CT of the zebrafish skeleton are powerful methods for revealing details of skeletal development, including the otoliths
Summary
The zebrafish, a species belonging to the Cyprinidae family, is a well-studied vertebrate developmental model because of its basal phylogenetic location, because of the high degree of homology between human and zebrafish genes [1] and organ systems [2] and because of the optical clarity of its embryos and larvae, allowing in vivo observations during development [3,4,5]. The ability to manipulate the embryo enables the use of different genetic. Zebrafish development: Micro-CT study design, data collection and analysis, decision to publish, or preparation of the manuscript
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