Visualization of basement membranes by a nidogen-based fluorescent reporter in mice

Q1 Medicine Matrix Biology Plus Pub Date : 2023-06-01 DOI:10.1016/j.mbplus.2023.100133
Sugiko Futaki , Ayano Horimoto , Chisei Shimono , Naoko Norioka , Yukimasa Taniguchi , Hitomi Hamaoka , Mari Kaneko , Mayo Shigeta , Takaya Abe , Kiyotoshi Sekiguchi , Yoichi Kondo
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引用次数: 3

Abstract

Basement membranes (BMs) are thin, sheet-like extracellular structures that cover the basal side of epithelial and endothelial tissues and provide structural and functional support to adjacent cell layers. The molecular structure of BMs is a fine meshwork that incorporates specialized extracellular matrix proteins. Recently, live visualization of BMs in invertebrates demonstrated that their structure is flexible and dynamically rearranged during cell differentiation and organogenesis. However, the BM dynamics in mammalian tissues remain to be elucidated. We developed a mammalian BM imaging probe based on nidogen-1, a major BM-specific protein. Recombinant human nidogen-1 fused with an enhanced green fluorescent protein (Nid1-EGFP) retains its ability to bind to other BM proteins, such as laminin, type IV collagen, and perlecan, in a solid-phase binding assay. When added to the culture medium of embryoid bodies derived from mouse ES cells, recombinant Nid1-EGFP accumulated in the BM zone of embryoid bodies, and BMs were visualized in vitro. For in vivo BM imaging, a knock-in reporter mouse line expressing human nidogen-1 fused to the red fluorescent protein mCherry (R26-CAG-Nid1-mCherry) was generated. R26-CAG-Nid1-mCherry showed fluorescently labeled BMs in early embryos and adult tissues, such as the epidermis, intestine, and skeletal muscles, whereas BM fluorescence was unclear in several other tissues, such as the lung and heart. In the retina, Nid1-mCherry fluorescence visualized the BMs of vascular endothelium and pericytes. In the developing retina, Nid1-mCherry fluorescence labeled the BM of the major central vessels; however, the BM fluorescence were hardly observed in the peripheral growing tips of the vascular network, despite the presence of endothelial BM. Time-lapse observation of the retinal vascular BM after photobleaching revealed gradual recovery of Nid1-mCherry fluorescence, suggesting the turnover of BM components in developing retinal blood vessels. To the best of our knowledge, this is the first demonstration of in vivo BM imaging using a genetically engineered mammalian model. Although R26-CAG-Nid1-mCherry has some limitations as an in vivo BM imaging model, it has potential applications in the study of BM dynamics during mammalian embryogenesis, tissue regeneration, and pathogenesis.

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基于氮源的荧光报告细胞对小鼠基底膜的可视化
基底膜(basal membrane, BMs)是覆盖上皮和内皮组织基底面的薄片状细胞外结构,为相邻的细胞层提供结构和功能支持。脑转移瘤的分子结构是一个包含特化细胞外基质蛋白的精细网络。近年来,无脊椎动物脑转移瘤的实时可视化研究表明,脑转移瘤的结构在细胞分化和器官发生过程中具有灵活性和动态重排性。然而,BM在哺乳动物组织中的动态仍有待阐明。我们开发了一种基于nidogen-1(一种主要的BM特异性蛋白)的哺乳动物BM成像探针。重组人nidogen-1与增强型绿色荧光蛋白(Nid1-EGFP)融合,在固相结合试验中保留了与其他BM蛋白(如层粘连蛋白、IV型胶原和perlecan)结合的能力。将重组Nid1-EGFP添加到小鼠胚胎干细胞衍生的胚状体培养基中,在胚状体的BM区积累,并在体外观察到BM。为了在体内进行BM成像,生成了表达人nidogen-1与红色荧光蛋白mCherry (R26-CAG-Nid1-mCherry)融合的敲入报告小鼠系。R26-CAG-Nid1-mCherry在早期胚胎和成人组织(如表皮、肠和骨骼肌)中显示有荧光标记的脑转移灶,而在其他一些组织(如肺和心脏)中,BM荧光不明显。在视网膜中,Nid1-mCherry荧光显示血管内皮和周细胞的脑转移。在发育中的视网膜中,Nid1-mCherry荧光标记主要中央血管的基底膜;然而,在血管网络的外周生长尖端几乎观察不到BM荧光,尽管内皮细胞存在BM。对光漂白后视网膜血管BM的延时观察显示,Nid1-mCherry荧光逐渐恢复,提示发育中的视网膜血管中BM成分发生了转换。据我们所知,这是第一次使用基因工程哺乳动物模型进行体内BM成像的演示。尽管R26-CAG-Nid1-mCherry作为活体脑转移成像模型存在一定的局限性,但它在哺乳动物胚胎发生、组织再生和发病机制中的脑转移动力学研究中具有潜在的应用价值。
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来源期刊
Matrix Biology Plus
Matrix Biology Plus Medicine-Histology
CiteScore
9.00
自引率
0.00%
发文量
25
审稿时长
105 days
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