{"title":"斑马鱼骨胶原动态的活体成像","authors":"Hiromu Hino, Shigeru Kondo, Junpei Kuroda","doi":"10.1016/j.bonr.2024.101748","DOIUrl":null,"url":null,"abstract":"<div><p>Type I collagen plays a pivotal role in shaping bone morphology and determining its physical properties by serving as a template for ossification. Nevertheless, the mechanisms underlying bone collagen formation, particularly the principles governing its orientation, remain unknown owing to the lack of a method that enables continuous in vivo observations. To address this challenge, we constructed a method to visualize bone collagen by tagging with green fluorescent protein (GFP) in zebrafish and observed the interactions between osteoblasts and collagen fibers during bone formation in vivo. When collagen type I alpha 2 chain (Col1a2)-GFP was expressed under the control of the osteoblast-specific promoters <em>osx</em> or <em>osc</em> in zebrafish, bone collagen was observed clearly enough to identify its localization, whereas collagen from other organs was not. Therefore, we determined that this method was of sufficient quality for the detailed in vivo observation of bone collagen. Next, bone collagen in the scales, fin rays, and opercular bones of zebrafish was observed in detail, when bone formation is more active. High-magnification imaging showed that Col1a2-GFP can visualize collagen sufficiently to analyze the collagen fiber orientation and microstructure of bones.</p><p>Furthermore, by simultaneously observation of bone collagen and osteoblasts, we successfully observed dynamic changes in the morphology and position of osteoblasts from the early stages of bone formation. It was also found that the localization pattern and orientation of bone collagen significantly differed depending on the choice of the expression promoter. Both promoters (<em>osx</em> and <em>osc</em>) used in this study are osteoblast-specific, but their Col1a2-GFP localizing regions within the bone were exclusive, with <em>osx</em> region localizing mainly to the outer edge of the bone and <em>osc</em> region localizing to the central area of the bone. 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High-magnification imaging showed that Col1a2-GFP can visualize collagen sufficiently to analyze the collagen fiber orientation and microstructure of bones.</p><p>Furthermore, by simultaneously observation of bone collagen and osteoblasts, we successfully observed dynamic changes in the morphology and position of osteoblasts from the early stages of bone formation. It was also found that the localization pattern and orientation of bone collagen significantly differed depending on the choice of the expression promoter. Both promoters (<em>osx</em> and <em>osc</em>) used in this study are osteoblast-specific, but their Col1a2-GFP localizing regions within the bone were exclusive, with <em>osx</em> region localizing mainly to the outer edge of the bone and <em>osc</em> region localizing to the central area of the bone. 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引用次数: 0
摘要
I 型胶原蛋白作为骨化的模板,在塑造骨骼形态和决定其物理特性方面发挥着关键作用。然而,由于缺乏可在体内进行连续观察的方法,骨胶原蛋白的形成机制,尤其是其定向原理仍不为人知。为了应对这一挑战,我们在斑马鱼体内构建了一种通过标记绿色荧光蛋白(GFP)来观察骨胶原蛋白的方法,并观察了骨形成过程中成骨细胞与胶原纤维之间的相互作用。在成骨细胞特异性启动子osx或osc的控制下,在斑马鱼体内表达Ⅰ型α2链胶原蛋白(Col1a2)-GFP时,可以清晰地观察到骨胶原蛋白,从而确定其定位,而其他器官的胶原蛋白则不能。因此,我们认为这种方法足以在体内详细观察骨胶原。接下来,我们详细观察了斑马鱼鳞片、鳍条和厣骨中的骨胶原,此时骨形成更为活跃。此外,通过同时观察骨胶原和成骨细胞,我们成功地从骨形成的早期阶段观察到了成骨细胞形态和位置的动态变化。我们还发现,骨胶原蛋白的定位模式和取向因表达启动子的选择而显著不同。本研究中使用的两种启动子(osx 和 osc)都具有成骨细胞特异性,但它们的 Col1a2-GFP 在骨中的定位区域却具有排他性,osx 区域主要定位在骨的外缘,而 osc 区域则定位在骨的中央区域。这些发现有助于更好地理解成骨细胞形成骨胶原的机制。
In vivo imaging of bone collagen dynamics in zebrafish
Type I collagen plays a pivotal role in shaping bone morphology and determining its physical properties by serving as a template for ossification. Nevertheless, the mechanisms underlying bone collagen formation, particularly the principles governing its orientation, remain unknown owing to the lack of a method that enables continuous in vivo observations. To address this challenge, we constructed a method to visualize bone collagen by tagging with green fluorescent protein (GFP) in zebrafish and observed the interactions between osteoblasts and collagen fibers during bone formation in vivo. When collagen type I alpha 2 chain (Col1a2)-GFP was expressed under the control of the osteoblast-specific promoters osx or osc in zebrafish, bone collagen was observed clearly enough to identify its localization, whereas collagen from other organs was not. Therefore, we determined that this method was of sufficient quality for the detailed in vivo observation of bone collagen. Next, bone collagen in the scales, fin rays, and opercular bones of zebrafish was observed in detail, when bone formation is more active. High-magnification imaging showed that Col1a2-GFP can visualize collagen sufficiently to analyze the collagen fiber orientation and microstructure of bones.
Furthermore, by simultaneously observation of bone collagen and osteoblasts, we successfully observed dynamic changes in the morphology and position of osteoblasts from the early stages of bone formation. It was also found that the localization pattern and orientation of bone collagen significantly differed depending on the choice of the expression promoter. Both promoters (osx and osc) used in this study are osteoblast-specific, but their Col1a2-GFP localizing regions within the bone were exclusive, with osx region localizing mainly to the outer edge of the bone and osc region localizing to the central area of the bone. This suggests the existence of distinct osteoblast subpopulations with different gene expression profiles, each of which may play a unique role in osteogenesis.
These findings would contribute to a better understanding of the mechanisms governing bone collagen formation by osteoblasts.
Bone ReportsMedicine-Orthopedics and Sports Medicine
CiteScore
4.30
自引率
4.00%
发文量
444
审稿时长
57 days
期刊介绍:
Bone Reports is an interdisciplinary forum for the rapid publication of Original Research Articles and Case Reports across basic, translational and clinical aspects of bone and mineral metabolism. The journal publishes papers that are scientifically sound, with the peer review process focused principally on verifying sound methodologies, and correct data analysis and interpretation. We welcome studies either replicating or failing to replicate a previous study, and null findings. We fulfil a critical and current need to enhance research by publishing reproducibility studies and null findings.