Zebrafish skeleton development: High resolution micro-CT and FIB-SEM block surface serial imaging for phenotype identification

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.尽管骨骼是当前研究最为深入的活体生物材料之一，但关于骨骼形成的具体机制仍存在诸多未解之谜，其中涵盖发育过程中骨骼结构的精细细节。本研究采用钙黄绿素（calcein）荧光标记、高分辨率显微计算机断层扫描（micro-CT）三维成像技术，以及块面串行成像模式下的聚焦离子束-扫描电子显微镜（FIB-SEM），对斑马鱼幼体的骨骼发育过程开展了监测。针对受精后前30天的胚胎，我们对比了野生型与透明斑马鱼nacre突变体的骨骼钙黄绿素染色结果与显微CT成像数据，发现二者存在显著差异。我们对发育过程中包括耳石（otolith）在内的骨骼体积与矿物质含量进行了定量分析，结果证实，包括耳石发育在内的这类发育差异可用于表型鉴定。此外，高分辨率成像结果显示，在轴向骨骼的首块骨骼形成之前，脊索（notochord）中已存在矿化聚集体，这类结构或参与矿物质的储存过程。本研究结果凸显了上述高分辨率三维成像技术在斑马鱼骨骼表征中的应用潜力，相关成果也将为深入解析骨骼发育机制及骨骼表型表征提供极具价值的参考依据。