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

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...

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Autores principales: Futaki, Sugiko, Horimoto, Ayano, Shimono, Chisei, Norioka, Naoko, Taniguchi, Yukimasa, Hamaoka, Hitomi, Kaneko, Mari, Shigeta, Mayo, Abe, Takaya, Sekiguchi, Kiyotoshi, Kondo, Yoichi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10149278/
https://www.ncbi.nlm.nih.gov/pubmed/37131404
http://dx.doi.org/10.1016/j.mbplus.2023.100133
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author Futaki, Sugiko
Horimoto, Ayano
Shimono, Chisei
Norioka, Naoko
Taniguchi, Yukimasa
Hamaoka, Hitomi
Kaneko, Mari
Shigeta, Mayo
Abe, Takaya
Sekiguchi, Kiyotoshi
Kondo, Yoichi
author_facet Futaki, Sugiko
Horimoto, Ayano
Shimono, Chisei
Norioka, Naoko
Taniguchi, Yukimasa
Hamaoka, Hitomi
Kaneko, Mari
Shigeta, Mayo
Abe, Takaya
Sekiguchi, Kiyotoshi
Kondo, Yoichi
author_sort Futaki, Sugiko
collection PubMed
description 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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spelling pubmed-101492782023-05-01 Visualization of basement membranes by a nidogen-based fluorescent reporter in mice Futaki, Sugiko Horimoto, Ayano Shimono, Chisei Norioka, Naoko Taniguchi, Yukimasa Hamaoka, Hitomi Kaneko, Mari Shigeta, Mayo Abe, Takaya Sekiguchi, Kiyotoshi Kondo, Yoichi Matrix Biol Plus Research Article 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. Elsevier 2023-04-08 /pmc/articles/PMC10149278/ /pubmed/37131404 http://dx.doi.org/10.1016/j.mbplus.2023.100133 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Futaki, Sugiko
Horimoto, Ayano
Shimono, Chisei
Norioka, Naoko
Taniguchi, Yukimasa
Hamaoka, Hitomi
Kaneko, Mari
Shigeta, Mayo
Abe, Takaya
Sekiguchi, Kiyotoshi
Kondo, Yoichi
Visualization of basement membranes by a nidogen-based fluorescent reporter in mice
title Visualization of basement membranes by a nidogen-based fluorescent reporter in mice
title_full Visualization of basement membranes by a nidogen-based fluorescent reporter in mice
title_fullStr Visualization of basement membranes by a nidogen-based fluorescent reporter in mice
title_full_unstemmed Visualization of basement membranes by a nidogen-based fluorescent reporter in mice
title_short Visualization of basement membranes by a nidogen-based fluorescent reporter in mice
title_sort visualization of basement membranes by a nidogen-based fluorescent reporter in mice
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10149278/
https://www.ncbi.nlm.nih.gov/pubmed/37131404
http://dx.doi.org/10.1016/j.mbplus.2023.100133
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