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.

Munenori Habuta,Seiichi Oyadomari,Ayuko Takayama,Hideyo Ohuchi,Tetsuya Bando,Satoru Miyaishi,Hitomi Kono,Akihiro Yasue,Keita Sato,Ken-Ichi T Suzuki,Eiji Tanaka,Hirofumi Fujita,Harald Ehrhardt

doi:10.1371/journal.pone.0240333

Abstract

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.

Highlights

The recently developed CRISPR/Cas9 system provides a highly efficient means for editing the genomes of model and non-model organisms
According to Hashimoto and Takemoto (2015) [15], the resultant Fibroblast growth factor 10 (Fgf10)-CRISPR F0 embryos were classified by the limb phenotype into three types: three limb phenotypes: no limb (type I), no limb; type II, limb defect; and type III, normal limbs
Fgf10 genome edited F0 mice are classified into three limb phenotypes: no limb, limb defect, and normal limbs

Summary

Introduction

The recently developed CRISPR/Cas system provides a highly efficient means for editing the genomes of model and non-model organisms. CRISPR/ Cas promises the possibility of an ultimate cure for genetic diseases by enabling replacement of mutated genes with normal alleles (reviewed in [3]); genome-edited founder mice often exhibit somatic mosaicism in the targeted gene, meaning more than two mutated alleles for that gene are mixed in the same mouse [4,5,6]. Such mosaicism may be undesirable when it complicates phenotypic analysis [7]. Recent DNA sequencing advances have enabled us to identify genetic mosaicism even in phenotypically normal individuals, the pathological significance of which is still unclear (reviewed in [9, 10])

Methods

Results

Conclusion