Dharmadhikari Avinash V, Sun Jenny J, Gogolewski Krzysztof, Carofino Brandi L, Ustiyan Vladimir, Hill Misty, Majewski Tadeusz, Szafranski Przemyslaw, Justice Monica J, Ray Russell S, Dickinson Mary E, Kalinichenko Vladimir V, Gambin Anna, Stankiewicz Paweł
Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Program in Translational Biology & Molecular Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Biol Open. 2016 Nov 15;5(11):1595-1606. doi: 10.1242/bio.019208.
FOXF1 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.
在患有新生儿致死性肺发育障碍——肺泡毛细血管发育不良伴肺静脉错位（ACDMPV）的新生儿中，已报道存在FOXF1杂合点突变和基因组缺失。然而，在任何人类疾病状态下，尚未鉴定出FOXF1的功能获得性突变。为了研究FOXF1过表达在肺发育中的作用，我们通过将一个Cre诱导型Foxf1等位基因敲入ROSA26（R26）位点，构建了一个Foxf1过表达小鼠模型。使用微型计算机断层扫描（micro-CT）、头出式体积描记法、ChIP-seq和转录组分析、免疫组织化学以及肺组织病理学对小鼠进行表型分析。当与Tie2-cre小鼠杂交时，35%的杂合R26-Lox-Stop-Lox（LSL）-Foxf1胚胎期（E）15.5胚胎表现出皮下水肿、出血，并在围产期死亡，Tie2-cre可使Foxf1在内皮细胞和造血细胞中过表达。组织病理学和micro-CT评估显示，R26Foxf1; Tie2-cre胚胎的肺不成熟，血管网络减少。详细的体积描记法研究证实，新生小鼠存在呼吸功能缺陷。对E18.5肺组织进行的ChIP-seq和转录组分析确定Sox11、Ghr、Ednrb和Slit2为FOXF1的潜在下游靶点。我们的研究表明，高度剂量敏感的Foxf1过表达会损害肺发育并导致血管异常。在考虑潜在的基因治疗方法来治疗FOXF1剂量异常的疾病（如ACDMPV）时，这具有重要的临床意义。
