Yu Tingsheng, Winkler Christoph
Department of Biological Sciences, National University of Singapore; NUS Centre for Bioimaging Sciences (CBIS), National University of Singapore.
Department of Biological Sciences, National University of Singapore; NUS Centre for Bioimaging Sciences (CBIS), National University of Singapore;
J Vis Exp. 2017 Jan 1(119):55025. doi: 10.3791/55025.
Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor κB Ligand (RANKL) under the control of a heat shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.
形成骨的成骨细胞与吸收骨的破骨细胞相互作用，以协调骨基质的更新并控制骨骼稳态。青鳉和斑马鱼幼体被广泛用于分析骨形成、退化和修复过程中骨细胞的行为。它们的光学透明性使得能够可视化荧光标记的骨细胞以及与矿化骨骼基质结合的荧光染料。我们实验室已培育出转基因青鳉鱼，其在热休克诱导型启动子的控制下表达破骨细胞诱导因子核因子κB受体活化因子配体（RANKL）。RANKL的异位表达导致活化破骨细胞过度形成，这在组织蛋白酶K（ctsk）启动子控制下表达nlGFP的报告基因品系中可以观察到。RANKL诱导和异位破骨细胞形成会导致严重的骨质疏松样表型。在破骨细胞中表达ctsk:nlGFP以及在早熟成骨细胞中在osterix（osx）启动子控制下表达mCherry的复合转基因青鳉品系，可用于研究这两种细胞类型的相互作用。这有助于在骨退化和修复条件下对细胞行为进行体内观察。在这里，我们描述了使用该系统测试一种常用于人类骨质疏松治疗的药物，并描述了一种活体成像方案。青鳉模型补充了细胞培养和小鼠研究，并为骨骼系统中药物作用的体内分析提供了一个新系统。
