Lethal lung hypoplasia and vascular defects in mice with conditional Foxf1 overexpression.

Avinash V Dharmadhikari,Jenny J Sun,Misty Hill,Mary E Dickinson,Russell S Ray,Monica J Justice,Przemyslaw Szafranski,Anna Gambin,Tadeusz Majewski,Brandi L Carofino,Paweł Stankiewicz,Vladimir V Kalinichenko,Krzysztof Gogolewski,Vladimir Ustiyan

doi:10.1242/bio.019208

Abstract

ABSTRACTFOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with the neonatally lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in any human disease conditions. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking-in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 embryonic day (E)15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage-sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.

Highlights

The FOXF1 (Forkhead box F1) gene located on chromosome 16q24.1 encodes a member of the FOX family of transcription factors characterized by a distinct forkhead DNA binding domain, and plays an important role in epithelium-mesenchyme signaling as a downstream target of Sonic hedgehog (Mahlapuu et al 2001; Dharmadhikari et al 2015)
We tested overexpression of Foxf1 in all tissues by crossing the R26-LSL-Foxf1 and the CMV-cre lines. We found that it led to embryonic lethality of R26Foxf1; CMV-cre embryos, displaying hemorrhage at E12.5 (Fig. S2)
Quantitative RT-PCR analysis showed that Foxf1 was overexpressed 1.7 fold in R26Foxf1; Tie2-cre E18.5 lungs compared to R26-LSL-Foxf1 control lungs (Fig. 1A). qRT-PCR on RNA isolated from flow sorted pulmonary endothelial cells (n=2-3 lungs) showed a trend towards overexpression of Foxf1 compared to wildtype littermates (Fig S3)

Summary

Introduction

The FOXF1 (Forkhead box F1) gene located on chromosome 16q24.1 encodes a member of the FOX family of transcription factors characterized by a distinct forkhead DNA binding domain, and plays an important role in epithelium-mesenchyme signaling as a downstream target of Sonic hedgehog (Mahlapuu et al 2001; Dharmadhikari et al 2015). In the mouse embryonic lungs, Foxf expression is restricted to mesenchyme-derived cells such as alveolar endothelial cells and peribronchiolar smooth muscle cells (Kalinichenko et al 2001a). Heterozygous point mutations and genomic deletions involving FOXF1 or its upstream enhancer have been reported in newborns with a lethal lung developmental disorder Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV, OMIM 265380) with or without defects involving heart, gastrointestinal, or genitourinary systems (Stankiewicz et al 2009; Bishop et al 2011; Sen et al 2013). Majority of Foxf1+/- mice die perinatally, exhibiting defects in lung vasculature, similar to those in patients with ACDMPV (Kalinichenko et al 2001b; Stankiewicz et al 2009)

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Results

Conclusion