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Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung

CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective...

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Autores principales: Habuta, Munenori, Yasue, Akihiro, Suzuki, Ken-ichi T., Fujita, Hirofumi, Sato, Keita, Kono, Hitomi, Takayama, Ayuko, Bando, Tetsuya, Miyaishi, Satoru, Oyadomari, Seiichi, Tanaka, Eiji, Ohuchi, Hideyo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561199/
https://www.ncbi.nlm.nih.gov/pubmed/33057360
http://dx.doi.org/10.1371/journal.pone.0240333
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author Habuta, Munenori
Yasue, Akihiro
Suzuki, Ken-ichi T.
Fujita, Hirofumi
Sato, Keita
Kono, Hitomi
Takayama, Ayuko
Bando, Tetsuya
Miyaishi, Satoru
Oyadomari, Seiichi
Tanaka, Eiji
Ohuchi, Hideyo
author_facet Habuta, Munenori
Yasue, Akihiro
Suzuki, Ken-ichi T.
Fujita, Hirofumi
Sato, Keita
Kono, Hitomi
Takayama, Ayuko
Bando, Tetsuya
Miyaishi, Satoru
Oyadomari, Seiichi
Tanaka, Eiji
Ohuchi, Hideyo
author_sort Habuta, Munenori
collection PubMed
description CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly, according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in type III (mean ± standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functional Fgf10 genotypes. These data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR F0 mouse would present an ideal experimental system to explore it.
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spelling pubmed-75611992020-10-21 Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung Habuta, Munenori Yasue, Akihiro Suzuki, Ken-ichi T. Fujita, Hirofumi Sato, Keita Kono, Hitomi Takayama, Ayuko Bando, Tetsuya Miyaishi, Satoru Oyadomari, Seiichi Tanaka, Eiji Ohuchi, Hideyo PLoS One Research Article CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly, according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in type III (mean ± standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functional Fgf10 genotypes. These data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR F0 mouse would present an ideal experimental system to explore it. Public Library of Science 2020-10-15 /pmc/articles/PMC7561199/ /pubmed/33057360 http://dx.doi.org/10.1371/journal.pone.0240333 Text en © 2020 Habuta et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Habuta, Munenori
Yasue, Akihiro
Suzuki, Ken-ichi T.
Fujita, Hirofumi
Sato, Keita
Kono, Hitomi
Takayama, Ayuko
Bando, Tetsuya
Miyaishi, Satoru
Oyadomari, Seiichi
Tanaka, Eiji
Ohuchi, Hideyo
Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung
title Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung
title_full Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung
title_fullStr Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung
title_full_unstemmed Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung
title_short Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung
title_sort fgf10-crispr mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561199/
https://www.ncbi.nlm.nih.gov/pubmed/33057360
http://dx.doi.org/10.1371/journal.pone.0240333
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