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. 2014 Sep 26;115(8):709-20.
doi: 10.1161/CIRCRESAHA.115.304382. Epub 2014 Aug 4.

FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells

Xiaomeng Ren  1 , Vladimir Ustiyan  1 , Arun Pradhan  1 , Yuqi Cai  1 , Jamie A Havrilak  1 , Craig S Bolte  1 , John M Shannon  1 , Tanya V Kalin  1 , Vladimir V Kalinichenko  2
Affiliations

FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells

Xiaomeng Ren et al. Circ Res. .

Abstract

Rationale: Inactivating mutations in the Forkhead Box transcription factor F1 (FOXF1) gene locus are frequently found in patients with alveolar capillary dysplasia with misalignment of pulmonary veins, a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardiovascular, and gastrointestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal, and gall bladder morphogenesis.

Objective: Although FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and patients with alveolar capillary dysplasia with misalignment of pulmonary veins remain uncharacterized because of lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach.

Methods and results: A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia, and vascular abnormalities in the lung, placenta, yolk sac, and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited vascular endothelial growth factor signaling, and decreased expression of endothelial genes critical for vascular development, including vascular endothelial growth factor receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin β3, ephrin B2, Tie2, and the noncoding RNA Fendrr. Chromatin immunoprecipitation assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1, and Tie2 genes are direct transcriptional targets of FOXF1.

Conclusions: FOXF1 is required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling.

Keywords: developmental biology; endothelial cells; pulmonary circulation; vascular endothelial growth factor A.

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Figures

Figure 1
Figure 1. FOXF1 is expressed in mesenchymal and endothelial cells of the developing embryo
(A–I) Immunostaining shows that FOXF1 is expressed in mesenchyme-derived cells in E13.5 wild type mouse embryos (A). FOXF1 is detected in cartilage primordiums of vertebral bodies (CPVB in B), oral cavity (Ca in C), tongue (To in D), atrioventricular cardiac cushions (CC in E), trachea (Tr) and esophagus (Es) (F), lung (Lu in G), small intestine (Si in H), stomach (St in I). (J–O) FOXF1 is expressed in endothelial cells. Low levels of FOXF1 were detected in a subset of endothelial cells (shown with arrows) located in the inferior vena cava (J) and pulmonary vein (K). FOXF1 is detected in blood vessels of the yolk sac (L and O), lung (N) and the fetal part of the placenta (M). A composite of four different images is shown in O. FOXF1 protein was not found in hematopoietic cells. (P–R) FOXF1 co-localizes with endothelial markers Flk1 (P), Isolectin B4 (Q) and vWF (R) in E12.5 lungs. Location of FOXF1 in nuclei of endothelial cells is shown with white arrows. Autofluorescence of red blood cells is denoted with *. Abbreviations: ep, epithelial cells; me, mesenchyme; Ve, blood vessel; AG, adrenal gland; He, heart; Ki, kidney; Li, liver; LJ, lower jaw; UJ, upper jaw; Sp, spleen; Vb, vertebrae. Scale bars: A, 100µm; B–M, 50µm; N–O, 20µm; P–R, 10µm.
Figure 2
Figure 2. Generation of Foxf1fl/fl mice and conditional deletion of FOXF1 from endothelial cells
(A) Schematic diagram of Foxf1 gene-targeting construct with two Frt sites (white arrows) and three LoxP sites (black arrowheads) that surround the Neomycin (neo) gene and exon 1 (E1), encoding the DNA-binding domain of the FOXF1 protein. The Neo cassette was removed after breeding of Foxf1-floxed mice with β-actin (ACT)-FLP1 mice. Endothelial deletion of FOXF1 was achieved by breeding with Tie2-Cre mice. (B) PCR of mouse tail DNA using primers (P1–P7). Locations of primers are indicated in panel A. (C) β-gal activity (blue staining) is detected in both endothelial and hematopoietic cells in the yolk sac of Tie2-Cre Foxf1fl/fl /R26R E8.5 embryos. Slides were counterstained with nuclear fast red (red nuclei). Inserts show blood vessels in the yolk sac (top insert) and embryo proper (bottom insert). (D) Immunostaining shows the presence of FOXF1 protein in endothelial cells (black arrowheads) of control Foxf1fl/fl embryos. FOXF1 staining is absent from majority of endothelial cells of Tie2-Cre Foxf1fl/fl embryos (white arrowheads). Abbreviations: Br, bronchiole; Ve, blood vessel. Scale bars are 50µm.
Figure 3
Figure 3. Embryonic abnormalities in Tie2-Cre Foxf1fl/fl embryos
(A) Tie2-Cre Foxf1fl/fl embryos (KO) exhibit growth retardation and severe polyhydramnios (white arrowheads) at E13.5–14.5. Foxf1fl/fl littermates (fl/fl) are shown for comparison. Boundary of the liver is indicated by blue line. Pericardial efflux is shown with a black arrow on H&E-stained section of Tie2-Cre Foxf1fl/fl embryos. Body weight is decreased in Tie2-Cre Foxf1fl/fl E13.5 embryos (n=10) compared to control Foxf1fl/fl littermates (n=43). (B) Liver size was decreased in Tie2-Cre Foxf1fl/fl embryos. Occipital frontal diameter (OFD) of the liver and the ratio between OFD and the Crown rump length (CRL) were significantly reduced after deletion of FOXF1 (n=5). (C–D) Diminished vascular branching in the yolk sac of FOXF1 deficient embryos. The whole mount immunoistaining was performed using endomucin Abs. Confocal microscopy was used to quantitate the vessel density as a ratio between endothelial (endomucin) and epithelial (E-cadherin) staining (bottom panel in D). p<0.05 is *, p<0.01 is **. Abbreviations: Ht, heart; Li, liver; Lu, lung. Scale bars: A, 50µm; B, 1mm; C, 50µm, D, 100µm.
Figure 4
Figure 4. Altered expression of endothelial genes in the yolk sac and lung of Tie2-Cre Foxf1fl/fl embryos
(A–B) Total RNA was prepared from yolk sacs of Tie2-Cre Foxf1fl/fl (KO) and control Tie2-Cre Foxf1fl/+ E12.5 embryos and analyzed by qRT-PCR. Decreased mRNAs of Foxf1, Flt-1, Flk-1 and Angpt 1 were found in Tie2-Cre Foxf1fl/fl yolk sacs. Angpt2 and Nrp1 mRNAs were increased (n=5). Expression levels were normalized to β-actin mRNA. (C) Immunohistochemical staining shows reduced PECAM1 and SOX-17 in lungs of FOXF1 KO embryos. The intensity of Flk1 staining was also reduced in FOXF1 KO lungs. (D) Decreased mRNAs of Pecam1, Sox-17, CD34, Flt1, Flk1 and Pdgfb were found in whole lung RNA from E12.5 KO embryos (n=6). p<0.05 is *, p<0.01 is **. Scale bars are 50µm.
Figure 5
Figure 5. Reduced vascular branching in Pdgfb-CreER Foxf1fl/fl embryos
(A) Pdgfb-CreER Foxf1fl/fl embryos were treated with tamoxifen at E9.5 and harvested at E12.5. Polyhydramnios in FOXF1-deficient embryos is indicated with white arrowhead. PCR of genomic tail DNA shows Cre-mediated recombination. Flow cytometry shows GFP fluorescence, which is associated with the Pdgfb-CreER transgene, in 60% of Pecam1+ endothelial cells (EC) isolated from the yolk sac. GFP is not detected in a majority of CD45+ hematopoietic cells (HC). (B) Increased β-gal activity and decreased FOXF1 staining is observed in lungs of Pdgfb-CreER Foxf1fl/fl/ R26R E17.5 embryos. (C) Immunostaining for Pecam1 and endomucin shows reduced vessel (Ve) density in the placenta and yolk sac of FOXF1-deficient embryos. Quantification is shown in bottom panel. (D) Decreased numbers of Pecam1+ endothelial cells are found in FOXF1-deficient lungs by flow cytometry (n=5). p<0.05 is *, p<0.01 is **. Scale bars are 50µm.
Figure 6
Figure 6. siRNA-mediated depletion of FOXF1 impairs angiogenesis in matrigel and inhibits VEGF signaling in cultured MFLM-91U cells
(A) MFLM-91U cells were transfected with either FOXF1 siRNA or control non-targeting siRNA. Forty-eight hours after siRNA transfection, matrigel angiogenesis assay was performed in the presence and absence of VEGF-A(165). FOXF1 knockdown reduced the number and total length of endothelial sprouts (n=5). (B) qRT-PCR shows reduced Foxf1, Flk1 and Pdgfb mRNAs at 24 hrs after siRNA transfection. Angp2 mRNA was increased after depletion of FOXF1. (C) Altered protein levels of FOXF1, Flk1, Flt1, PECAM-1, PDGFb and Angpt2 at 48 hrs after siRNA transfection are shown by Western blots. (D) FOXF1-depleted MFLM-91U cells are resistant to VEGF-A(165) stimulation as indicated by decreases in phosphorylated Erk1/2 (pErk1/2) and phosphorylated Akt (pAkt). Quantification of Western blots is shown in right panels in C and D. p<0.05 is *, p<0.01 is **. Scale bars are 500µm.
Figure 7
Figure 7. FOXF1 directly regulates expression of endothelial genes critical for angiogenesis and VEGF signaling
(A) Reduced BrdU incorporation and increased annexin V staining is detected by flow cytometry in endothelial cells of Tie2-Cre Foxf1fl/fl E12.5 embryos. The dotted line represents non-specific staining with BrdU Abs. (B) GFP and tdTomato fluorescence were measured in endothelial (CD45−PECAM-1+) and hematopoietic cells (CD45+PECAM-1−) isolated from Tie2-Cre Foxf1fl/fl mT/mG yolk sacs. GFP is not detected in cells from control Foxf1fl/fl mT/mG yolk sacs. Efficiency of Cre-mediated recombination is shown as a percentage of GFP+ cells among total cells isolated from the yolk sac (n=5) (right panel). BrdU incorporation is decreased in GFP+ endothelial cells at E10.5 (right middle panels). (C) Endothelial cells were flow sorted from E12.5 yolk sacs. qRT-PCR was used to examine mRNAs. (D) ChIP assay shows that FOXF1 binds to promoter regions of Pecam-1, Pdgfb, Tie-2, Flt1 and Flk1 genes. FOXF1 does not bind to Nrp1 and Angpt2 promoters. Fetal endothelial MFLM-91U cells were used for ChIP. p<0.05 is *, p<0.01 is **.
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Comment in

  • Fox(y) regulators of VEGF receptors.
    Ricard N, Simons M. Ricard N, et al. Circ Res. 2014 Sep 26;115(8):683-5. doi: 10.1161/CIRCRESAHA.114.304974. Circ Res. 2014. PMID: 25258399 Free PMC article. No abstract available.

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