One of the most frequently applied techniques in zebrafish (D. rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue- specific expression of frequently used fluorophores such as GFP or mCherry, is relatively easy with modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV-light, a process called photoconversion. Here, we illustrate the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV-light. First, we demonstrate that osx:Kaede shows an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, we show that combining two transgenics, osx:Kaede and osc:GFP with similar emission wavelengths is possible when using a pcFP such as Kaede. This article is protected by copyright. All rights reserved.

中文翻译：

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光转化荧光蛋白：斑马鱼骨骼成像的多功能工具

斑马鱼 (D. rerio) 研究中最常用的技术之一是使用转基因报告系对特定细胞群进行可视化或操作。这些转基因斑马鱼的生成，展示了常用荧光团（如 GFP 或 mCherry）的细胞或组织特异性表达，使用现代技术相对容易。可以在同一动物中同时监测具有不同发射波长和由不同启动子驱动的荧光团。光转换荧光蛋白 (pcFP) 与这些标准荧光团不同，因为它们的发射光谱在暴露于紫外线时会发生变化，这一过程称为光转换。这里，我们说明了在先前生成的 osx:Kaede 转基因品系中的斑马鱼骨骼研究中使用 pcFP 进行单和双荧光染料成像的好处和多功能性。在该品系中，Kaede 在 osterix（也称为 sp7）启动子的控制下表达，从而标记未成熟的成骨细胞，在紫外线照射下可以从绿色荧光转换为红色荧光。首先，我们证明 osx:Kaede 在幼虫和成虫阶段显示出类似于先前描述的 osx:nuGFP 转基因系的表达模式，从而验证该系用于未成熟成骨细胞的成像。更深入的实验突出了 osx:Kaede 的不同应用，例如谱系追踪及其与体内骨骼染色和其他转基因背景的结合使用。矿物染色结合 osx:Kaede 证实脊索的成骨细胞独立矿化。成骨细胞谱系追踪揭示了鳍再生过程中成骨细胞的迁移和去分化。最后，我们表明，当使用 pcFP（例如 Kaede）时，可以将具有相似发射波长的两个转基因 osx:Kaede 和 osc:GFP 结合使用。本文受版权保护。版权所有。