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Am J Physiol Lung Cell Mol Physiol 286: L521-L530, 2004. First published November 7, 2003; doi:10.1152/ajplung.00212.2003
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TRANSLATIONAL PHYSIOLOGY

Foxf1 haploinsufficiency reduces Notch-2 signaling during mouse lung development

Vladimir V. Kalinichenko,1 Galina A. Gusarova,1,2 Il-Man Kim,1 Brian Shin,1 Helena M. Yoder,1 Jean Clark,3 Alexander M. Sapozhnikov,2 Jeffrey A. Whitsett,3 and Robert H. Costa1

1Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60607-7170; 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; and 3Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039

Submitted 2 July 2003 ; accepted in final form 31 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The forkhead box (Fox) f1 transcription factor is expressed in the mouse splanchnic (visceral) mesoderm, which contributes to development of the liver, gallbladder, lung, and intestinal tract. Pulmonary hemorrhage and peripheral microvascular defects were found in approximately half of the newborn Foxf1(+/-) mice, which expressed low levels of lung Foxf1 mRNA [low-Foxf1(+/-) mice]. Microvascular development was normal in the surviving newborn high-Foxf1(+/-) mice, which compensated for pulmonary Foxf1 haploinsufficiency and expressed wild-type Foxf1 levels. To identify expression of genes regulated by Foxf1, we used Affymetrix microarrays to determine embryonic lung RNAs influenced by Foxf1 haploinsufficiency. Embryonic Foxf1(+/-) lungs exhibited diminished expression of hepatocyte growth factor receptor c-Met, myosin VI, the transcription factors SP-3, BMI-1, ATF-2, and glucocorticoid receptor, and cell cycle inhibitors p53, p21Cip1, retinoblastoma, and p107. Furthermore, Notch-2 signaling was decreased in embryonic Foxf1(+/-) lungs, as evidenced by significantly reduced levels of the Notch-2 receptor and the Notch-2 downstream target hairy enhancer of split-1. The severity of the Notch-2-signaling defect in 18-day postcoitus Foxf1(+/-) lungs correlated with Foxf1 mRNA levels. Disruption of pulmonary Notch-2 signaling continued in newborn low-Foxf1(+/-) mice, which died of lung hemorrhage and failed to compensate for Foxf1 haploinsufficiency. In contrast, in newborn high-Foxf1(+/-) lungs, Notch-2 signaling was restored to the level found in wild-type mice, which was associated with normal microvascular formation and survival. Foxf1 haploinsufficiency disrupted pulmonary expression of genes in the Notch-2-signaling pathway and resulted in abnormal development of lung microvasculature.

winged helix DNA-binding domain; Notch-2 receptor; microarray analysis; forkhead box transcription factor; hepatocyte nuclear factor/forkhead homolog-8; forkhead-related activator-1

DEVELOPMENT OF THE MOUSE LUNG begins at 9.5 days postcoitus (pc) as lung buds arise from the laryngotracheal groove of the foregut endoderm. The lung buds invade the splanchnic mesenchyme and undergo dichotomous branching regulated by mesenchymal-epithelial cell signaling, which induces cellular proliferation, migration, and differentiation (67). Branching morphogenesis of the lung depends on mesenchymal-epithelial interactions mediated by various signaling molecules that regulate the process of lung morphogenesis and induce expression of cell type-specific transcription factors (6, 8, 11, 67, 70). These pulmonary mesenchymal-epithelial signaling proteins include Sonic hedgehog (32), bone morphogenetic protein (BMP)-4 (2, 68), hepatocyte growth factor (HGF) (41), fibroblast growth factor (FGF)-10 (1, 55), and FGF-7 (37, 47, 60).

The membrane-spanning Notch receptors are stimulated by Jagged-1, Jagged-2, or Delta ligands, present on the surface of neighboring cells, causing activation of a-disintegrin and metalloprotease (ADAM)-10 and {gamma}-secretase proteases, which mediate proteolytic cleavage of the Notch cytoplasmic domain (3, 16, 30, 61). The cleaved Notch cytoplasmic domain undergoes nuclear translocation, where it functions as a transcriptional coactivator protein for the CBF1/RBPj{kappa} transcription factors, which stimulate expression of the transcriptional repressor hairy enhancer of split-1 (Hes-1) (17, 20, 21, 23). Notch-1-mediated activation of the Hes-1 transcriptional repressor inhibits expression of basic helix-loop-helix transcription factor mammalian achaete-scute homolog 1 (9), which is required for differentiation of the pulmonary neuroendocrine cells (19). Furthermore, protein expression studies demonstrate that Notch-1 and Jagged-1 proteins are expressed in endothelial cells of pulmonary vessels and microvasculature, suggesting that they are involved in Notch-1 signaling during lung vasculogenesis (58). Although Notch-2 is also expressed in lung mesenchyme (49), the role of Notch-2 in lung microvascular development is unknown.

The forkhead box (Fox) proteins are an extensive family of transcription factors that share homology in the winged helix/Forkhead DNA-binding domain (10, 28). Its members play important roles in regulating transcription of genes involved in cellular proliferation (62, 64-66), differentiation (8, 11, 13, 46, 72), and metabolic homeostasis (22, 40). Foxf1 expression begins during gastrulation at 6.5 days pc in extraembryonic mesoderm, allantois, and lateral mesoderm (35, 48). Consistent with this expression pattern, Foxf1(-/-) mouse embryos die by 8 days pc because of defects in extraembryonic mesoderm development (25, 35). Foxf1 expression continues in septum transversum and splanchnic (visceral) mesoderm, which are critical for the mesenchymal-epithelial induction of gut-derived organs such as liver, gallbladder, lung, stomach, and intestine (27, 36, 48). Haploinsufficiency of the Foxf1 gene caused a variety of developmental abnormalities in the lung, gallbladder, esophagus, and trachea (25, 27, 31, 34). Liver regeneration studies demonstrate that Foxf1(+/-) mice exhibit defective activation of stellate cells in response to CCl4 liver injury, which results in elevated hepatocyte apoptosis and diminished induction of Notch-2 receptor and interferon-inducible protein-10 expression (24). Perinatal lethality from pulmonary hemorrhage was found in approximately half of Foxf1(+/-) newborn mice, which displayed an 80% reduction in wild-type (WT) pulmonary Foxf1 levels [low-Foxf1(+/-) mice], instead of the 50% expression expected for heterozygous mice. Lung hemorrhage in newborn low-Foxf1(+/-) mice was associated with severe fusion of lung lobes and defects in development of peripheral lung saccules and microvasculature (25, 31, 34). These developmental defects were associated with reduced pulmonary expression of fetal lung kinase-1, platelet-endothelial cell adhesion molecule (PECAM)-1, BMP-4, lung Kruppel-like factor, and T-box (Tbx2-Tbx5) transcription factors with delayed expression of FGF-10 (25, 31). Interestingly, expression of these lung genes was unchanged in the 40% of newborn Foxf1(+/-) mice that had compensated for pulmonary Foxf1 expression and displayed WT levels of Foxf1 mRNA. These high-Foxf1(+/-) mice exhibited normal development of peripheral lung saccules and microvasculature and less severe fusions of lung lobes (25, 31).

To identify pulmonary genes whose altered expression is associated with defective lung formation in Foxf1(+/-) mice, we used cDNA probes prepared from 18-day pc lung RNA from WT or Foxf1(+/-) embryos to analyze Affymetrix gene microarrays. We found that Notch-2 signaling was disrupted in 18-day pc Foxf1(+/-) lungs, as evidenced by significantly reduced levels of the Notch-2 receptor and the Notch-2 downstream target Hes-1, but a more severe reduction in Notch-2 signaling was found in embryonic low-Foxf1(+/-) lungs. Disruption of Notch-2 signaling continued in newborn low-Foxf1(+/-) lungs and was associated with perinatal lung hemorrhage and lethality with severe defects in peripheral lung microvasculature. In contrast, restored Notch-2 signaling and normal lung microvascular development were associated with compensation for Foxf1 haploinsufficiency in newborn high-Foxf1(+/-) lungs. These studies indicated that Foxf1 regulates expression of genes in the Notch-2-signaling pathway required for normal development of peripheral lung microvasculature.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Foxf1(+/-) mice. The generation of mice heterozygous for the Foxf1-targeted null allele has been described previously (25). The Foxf1-targeted allele consisted of an in-frame insertion of the {beta}-galactosidase ({beta}-gal) gene, which deleted the Foxf1 winged helix DNA-binding domain sequences required for DNA recognition and nuclear localization (50). Expression from the Foxf1-targeted allele resulted in a fusion protein consisting of the first 20 amino acids of the Foxf1 coding region fused to the NH2 terminus of the {beta}-gal protein. The Foxf1(+/-) mice were bred into the Black Swiss mouse background for four generations. Embryonic heart tissue was used to prepare genomic DNA for genotyping of 15- and 18-day pc embryos using PCR analysis with primers specific to the {beta}-gal gene, as described previously (25). We used RNase protection assays (RPA) with lung RNA to subdivide Foxf1(+/-) embryos and mice into two classes on the basis of pulmonary Foxf1 mRNA levels, as described previously (25). The high-Foxf1(+/-) mice displayed a compensatory increase in pulmonary expression of Foxf1 mRNA to levels found in WT lungs. The low-Foxf1(+/-) mice exhibited 20-30% of WT pulmonary levels of Foxf1 mRNA, instead of the expected 50% expression levels found in heterozygous mice. We killed the newborn offspring within 24 h after birth to obtain newborn lungs from WT, high-Foxf1(+/-), and low-Foxf1(+/-) (i.e., those that exhibit perinatal lethality from pulmonary hemorrhage) mice. The dissected lung tissue was used for preparation of RNA and total protein extracts and for paraffin embedding, as described previously (25). We subjected newborn mice to an injection of 5-bromo-2'-deoxyuridine (BrdU, 50 mg/g body wt ip; Sigma) 2 h before harvesting lungs to monitor DNA replication by immunohistochemically staining for BrdU incorporation, as described previously (66, 71). DNA replication was determined in 18-day pc embryonic lungs by immunohistochemical staining of lung sections with a monoclonal antibody against proliferating cell nuclear antigen (clone PC10, Roche Molecular Biochemicals, Indianapolis, IN). We detected the antibody-antigen complex using horse anti-mouse antibody conjugated with alkaline phosphatase (Vector Laboratories, Burlingame, CA), as described previously (25).

Immunohistochemical staining, Western blot analysis, and electrophoretic mobility shift assays. Lung tissue was dissected from 15- and 18-day pc mouse embryos or newborn mice, embedded in paraffin, sectioned, and used for immunohistochemical staining with rabbit polyclonal antibody against Notch-2 (catalog no. 25-255, Santa Cruz Biotechnology). Antibody-antigen complexes were detected using biotinylated goat anti-rabbit antibody (Pharmingen, San Diego, CA), avidin-alkaline phosphatase complex, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium substrate (Vector Laboratories), as described previously (25). Staining with rat monoclonal PECAM-1 (clone MEC 13.3) and mouse monoclonal {alpha}-smooth muscle actin antibodies (clone 1A4; Sigma) was performed as described elsewhere (25). Total protein extracts were prepared from 18-day pc, newborn, or adult mouse lungs and subjected to Western blot analysis to measure protein levels of Foxf1, Notch-2, HGF, p21, retinoblastoma (Rb) protein, and Hes-1. The antibodies used in this Western blot analysis are as follows: rabbit polyclonal antibodies against Notch-2 (1:200 dilution), Foxf1 (24) (1:500 dilution), HGF (catalog no. H-145, Santa Cruz Biotechnology; 1:200 dilution), p21 (Oncogene Research Products; 1:200 dilution), p53 (Cell Signaling; 1:1,000 dilution), goat polyclonal Hes-1 (catalog no. H-20, Santa Cruz Biotechnology; 1:100 dilution), mouse monoclonal Rb protein (clone G3-245, Pharmingen; 1:500 dilution), and mouse monoclonal {beta}-actin (clone AC-15, Sigma; 1:3,000 dilution) antibodies. Detection of the immune complex was accomplished by using secondary antibodies directly conjugated with horseradish peroxidase followed by chemiluminescence (ECL Plus, Amersham-Pharmacia Biotech, Bucking-hamshire, UK) and autoradiography with X-ray film (Kodak). Quantitation of expression levels was determined with Tiff files from scanned films by using the BioMax 1D program (Kodak). Pulmonary {beta}-actin expression signal was used for normalization control between different protein samples.

Osteosarcoma U2OS cells (100-mm plate) were left untransfected or transiently transfected with 15 µg of cytomegalovirus (CMV)-Foxf1 expression vector using Fugene-6; 24 h after transfection, nuclear protein extracts were prepared as described previously (48, 53, 54, 59). Electrophoretic mobility shift assays (EMSA) were performed with 5'-end radioactively labeled double-stranded oligonucleotides and 5 µg of nuclear extracts prepared from untransfected or CMV-Foxf1-transfected U2OS cells using binding reaction conditions described previously (48, 53, 54, 59). The Foxf1 protein-DNA complex was separated from unbound labeled DNA using native polyacrylamide electrophoresis, and this protein-DNA complex was visualized by autoradiography. For DNA competitions or antibody reactions in EMSA, we added 500-fold molar excess of cold competitor double-stranded oligonucleotide to the binding reaction or we preincubated nuclear extracts with 1 µl of affinity-purified Foxf1 peptide antibody for 30 min at room temperature before adding the nuclear extract to the binding reaction, as described previously (24, 27, 51).

Affymetrix cDNA array analysis and RPA. Total mouse lung RNA was prepared from 18-day pc embryonic high-Foxf1(+/-) or low-Foxf1(+/-) mouse lungs or WT littermates using RNA-STAT-60 (Tel-Test B, Friendswood, TX). To avoid individual variations, we combined 10 µg of RNA from three distinct embryonic lungs [high-Foxf1(+/-), low-Foxf1(+/-), or WT]. Synthesis of embryonic mouse lung cDNAs with CyDye nucleotides (Cy3 and Cy5), hybridization of Affymetrix GeneChip mouse expression set 430A (repeated twice), scanning, and analysis of cDNA microarrays were performed by the Center of Bioinformatics at Children's Hospital Medical Center (Cincinnati, OH). In Table 1, we summarize our focus on characterization of 17 genes, the expression levels of which were altered more than threefold in Foxf1(+/-) lungs compared with WT lungs. To verify expression levels of these genes, we used RT-PCR analysis with RNA isolated from 18-day pc embryonic lung to amplify cDNA segments and cloned them into the pCRII plasmid, which allows synthesis of antisense RNA probes for RPA. The following sense and antisense primers were used for amplification: 5'-ccttgttctggcaatctgcg-3' and 5'-ggaatggaaacctgggatgg-3' (GATA-5), 5'-cgagacccctgtgagaaaaacc-3' and 5'-atgttggcagcgtcctggaatgtc-3' (Notch-2), 5'-gagcacagaataaagtgaggaaggg-3' and 5'-gaccagagttggatggaaacagc-3' (collagen 18A1), 5'-agagggatggactgacgaatgc-3' and 5'-aaggaagcaaactggacgacag-3' (BMI-1), 5'-tgatggggaatgacttgggc-3' and 5'-cgtttgtctctttacctggggacc-3' [glucocorticoid receptor (GR) 1], 5'-gccgacaaaaaaggaaagtgtg-3' and 5'-gcttctgtatgtggactgcttgg-3' (ATF-2), 5'-aacccccatccagaatgtcgtc-3' and 5'-tgttgtctttccaaacccctcc-3' (c-Met), 5'-cgttttacattgagccagcagagag-3' and 5'-gccagagttgtgcgttttttcc-3' (ADAM-10), and 5'-gacttttgtcccacttgaatccc-3' and 5'-tccattgctgtgctcttagcg-3' [zonula occludens-1 (ZO-1)].


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  		Table 1. Embryonic lung expression profile of Foxf1 (+/–) vs. WT mouse embryos at 18 days pc

 

RPA was performed with [32P]UTP-labeled antisense RNA synthesized from plasmid templates with the appropriate RNA polymerase, as previously described (26, 63). RNA probe hybridization, RNase ONE (Promega, Madison, WI) digestion, electrophoresis of RNA protected fragments, and autoradiography were performed as described previously (26, 63). Quantitation of expression levels was determined from scanned X-ray films by using the BioMax 1D program (Kodak). The cyclophilin or ribosomal protein L32 hybridization signals were used for normalization control between different lung RNA samples. Synthesis of antisense mouse Foxf1 and cyclophilin RNA probes was described previously (25, 26).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Microarray analysis identifies genes in which expression is altered in Foxf1(+/-) lungs. To identify genes in which expression is associated with defects in Foxf1(+/-) lung formation, we analyzed mouse Affymetrix microarrays that were hybridized with cDNA probes synthesized from 18-day pc lung RNA prepared from low-Foxf1(+/-), high-Foxf1(+/-), or WT mouse embryos. This analysis identified genes in which expression was significantly increased or decreased in Foxf1(+/-) lungs or increased or decreased only in low-Foxf1(+/-) lungs (Table 1). To obtain probes for the genes affected by Foxf1 haploinsufficiency and verify their expression levels, we designed suitable primers for RT-PCR using WT or Foxf1(+/-) lung RNA and cloned these fragments in vectors for synthesis of antisense RNA hybridization probes. RPA and/or Western blot analyses were used to determine Foxf1 mRNA and protein levels (Fig. 1) and verify changes in expression of several of the pulmonary genes identified by Affymetrix microarray analysis.



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  		Fig. 1. Verification of altered Foxf1(+/-) lung genes by RNase protection assay (RPA) and Western blot analysis. A: altered mRNA levels of Foxf1(+/-) lung genes. Total RNA isolated from low-Foxf1(+/-), high-Foxf1(+/-), or wild-type (WT) lungs at 18 days postcoitus (dpc) was subjected to RPA with probes for zonula occludens-1 (ZO-1, a tight junction protein), collagen 18A1 (Col 18A1), hepatocyte growth factor (HGF) receptor c-Met, cyclophilin, and transcription factors BMI-1, ATF-2, GATA-2, and glucocorticoid receptor (GR). Each individual sample was normalized to its corresponding cyclophilin level. Values are means ± SD. B: Western blot analysis demonstrates that Foxf1 protein levels are diminished in low-Foxf1(+/-) lungs and unchanged in high-Foxf1(+/-) lungs compared with WT lungs. C: HGF levels do not change in Foxf1(+/-) lungs. Total protein extracts isolated from low-Foxf1(+/-), high-Foxf1(+/-), or WT lungs (18 dpc) were subjected to Western blot analysis using antibodies against Foxf1, HGF, or {beta}-actin (loading control). Protein levels were normalized to {beta}-actin. Values are means ± SD.

 

Subsets of lung RNAs were altered in Foxf1(+/-) embryos. Increased expression of several genes was observed in lungs of Foxf1(+/-) embryos. This subset of genes included zinc finger transcription factor GATA-5 (Table 1, Fig. 1A) and the homeodomain Hoxa4 (Table 1), both of which are coexpressed with Foxf1 in lung mesenchyme (38, 45). Foxf1(+/-) lungs also exhibited increased levels of extracellular matrix metalloproteinase-12 (MMP-12) mRNA (Table 1), which is involved in tissue remodeling (56, 69). Interestingly, {alpha}1-collagen type XVIII expression was selectively increased in low-Foxf1(+/-) lungs (Fig. 1A), which display a perinatal lung hemorrhage phenotype (25). Furthermore, decreased expression of transcription factors BMI-1, ATF-2, SP-3, and GR mRNA and reduced levels of myosin VI mRNA were observed in Foxf1(+/-) lungs (Table 1, Fig. 1A). A 70% reduction in expression of the tight junction protein ZO-1 mRNA (Fig. 1A) was observed and is consistent with disruption of the epithelial-endothelial interfaces found in newborn low-Foxf1(+/-) lungs (25). Moreover, Foxf1(+/-) lungs exhibited significant decreases in expression of the HGF receptor c-Met mRNA (Fig. 1A). Because HGF levels were also significantly decreased in Foxf1(+/-) gallbladders (27), we used Western blot analysis to examine HGF protein levels in Foxf1(+/-) lungs and found no difference in HGF expression between Foxf1(+/-) and WT lungs (Fig. 1B).

Defective microvascular development in low-Foxf1(+/-) lungs is associated with diminished expression of genes in the Notch-2-signaling pathway. A 70% reduction in Notch-2 receptor mRNA was found in high- and low-Foxf1(+/-) lungs at 15 days pc (Fig. 2A), which is before the compensatory increase in Foxf1 mRNA levels that occurs in ~40% of Foxf1(+/-) lungs at 18 days pc (Fig. 2, A and B). By 18 days pc, a more severe decrease in expression of the Notch-2 receptor was found in low-Foxf1(+/-) lungs, correlating with reduced pulmonary levels of Foxf1 mRNA compared with high-Foxf1(+/-) lungs (Fig. 2B, Table 1). To determine the amount of Notch-2 signaling in embryonic Foxf1(+/-) lungs, we used Western blot analysis to measure protein levels of Hes-1, a transcriptional repressor activated by the Notch-2-signaling pathway (17, 20, 21, 23). Although Hes-1 protein levels were nearly undetectable in lung extracts from low-Foxf1(+/-) embryos, they were reduced by 80% in embryonic high-Foxf1(+/-) lungs compared with 18-day pc WT lungs (Fig. 2C). In contrast, a similar reduction in expression of the ADAM-10 Notch-2-activating metalloprotease was found in 18-day pc low- and high-Foxf1(+/-) lungs (Fig. 2B, Table 1). These studies demonstrated that Notch-2 signaling was more severely affected in low-Foxf1(+/-) lungs, correlating with low pulmonary levels of Foxf1 mRNA. Analysis of the cellular expression pattern of the Notch-2 receptor protein by immunohistochemical staining demonstrated that it was expressed in WT lung mesenchyme at 15 and 18 days pc (Fig. 3, A, C, and G), a finding consistent with previously reported expression studies (49). Although mesenchymal cells of WT and low-Foxf1(+/-) lungs exhibited similar PECAM-1 (Fig. 3, E and F) and {alpha}-smooth muscle actin staining (Fig. 3, I and J), expression of the Notch-2 receptor protein was decreased in low-Foxf1(+/-) lung mesenchyme compared with age-matched embryonic lung controls (Fig. 3, B, D, and H). These data suggested that the expression pattern of Notch-2 overlaps with that of Foxf1 in the developing mouse lung (25, 31, 36, 48).



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  		Fig. 2. Disruption of Notch-2 signaling in embryonic Foxf1(+/-) lungs. A and B: diminished expression of Notch-2 and disintegrin metalloprotease Kuzbanian (ADAM-10) in embryonic Foxf1(+/-) lungs. Total lung RNA was isolated from low-Foxf1(+/-), high-Foxf1(+/-), or WT embryos at 15 (A) or 18 (B) dpc. RPA was performed with probes for Notch-2 receptor and Notch-2-activating metalloprotease ADAM-10. C: embryonic Foxf1(+/-) lungs displayed reduced levels of the downstream target hairy enhancer of split-1 (Hes-1) protein. Total protein extracts isolated from low-Foxf1(+/-), high-Foxf1(+/-), or WT lungs (18 dpc) were subjected to Western blot analysis using antibodies against Hes-1 and {beta}-actin. Protein levels were normalized to {beta}-actin. Values are means ± SD.

 


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  		Fig. 3. Reduced Notch-2 receptor expression in mesenchyme of embryonic Foxf1(+/-) lungs. Microtome sections of paraffin-embedded embryonic lungs were prepared from WT or low-Foxf1(+/-) embryos (A-J) and used for immunohistochemical staining with Notch-2 (A-D, G, and H), platelet-endothelial cell adhesion molecule (PECAM-1, E and F), or {alpha}-smooth muscle actin ({alpha}-SM; I and J) antibodies. Notch-2 staining was also performed with WT or high-Foxf1(+/-) newborn mice (K and L). Notch-2 staining was diminished in lung mesenchyme (Me) surrounding epithelial cells (Ep) in 15-dpc low-Foxf1(+/-) embryos (B and D). PECAM-1 (E and F) and {alpha}-SM expression (I and J) was not altered in mesenchyme of Foxf1(+/-) embryonic lung. Although reduction of Notch-2 was also observed in 18-dpc low-Foxf1(+/-) lungs (H), Notch-2 staining was not affected in lungs from high-Foxf1(+/-) newborn mice (L). Br, bronchiole; Ar, artery. Scale bar, 20 µm.

 

To determine whether Foxf1 directly binds to the Notch-2 receptor promoter, we performed EMSA with nuclear protein extract from CMV-Foxf1-transfected U2OS cells and two distinct oligonucleotides that contain potential Foxf1-binding sites in the promoter region of the mouse Notch-2 receptor gene (Fig. 4C,nt -984/-1006 and nt -2813/-2786). We also included EMSA with HFH1 oligonucleotide 25, which was isolated by DNA site selection and found to bind to recombinant Foxf1 protein with high affinity (44, 48). These putative Foxf1-binding sites in the Notch-2 promoter formed a Foxf1 protein-DNA complex similar to that with the high-affinity Foxf1 HFH1 oligonucleotide 25 binding site. These oligonucleotides formed specific complexes with Foxf1 protein, as demonstrated by the ability of Foxf1 antibody or cold competitor oligonucleotides to interfere with formation of the Foxf1 protein-DNA complexes (Fig. 4D). This result shows that Foxf1 directly binds to the Notch-2 receptor promoter region and that Foxf1 protein may therefore regulate transcription of the Notch-2 receptor gene.



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  		Fig. 4. Diminished Notch-2 expression is associated with lung hemorrhage in newborn low-Foxf1(+/-) mice. A: diminished Notch-2 receptor expression in low-Foxf1(+/-) newborn mice. Total lung RNA was isolated from newborn WT, low-Foxf1(+/-), and high-Foxf1(+/-) mice and used for RPA with probes for Notch-2 receptor or ADAM-10. Each individual sample was normalized to its corresponding cyclophilin or L32 level. Values are means ± SD. B: high-Foxf1(+/-) lungs displayed restored protein levels of Notch-2 receptor and Hes-1. Total protein extracts isolated from newborn high-Foxf1(+/-) or WT lungs were subjected to Western blot analysis using antibodies against Notch-2, Hes-1, or {beta}-actin (loading control). Protein levels were normalized to {beta}-actin. Values are means ± SD. C: potential Foxf1-binding sequences in the Notch-2 receptor promoter region at -984/-1,006 and -2,813/-2,786 bp upstream of start site. Also shown is the high-affinity Foxf1 DNA target sequence isolated by site selection HFH1 oligonucleotide 25 and Foxf1 consensus binding sequence, as described previously (48). V is not "T," N is any nucleotide, D is not "C," R is "A" or "G," and Y is "C" or "T." D: EMSA shows that Foxf1 binds to potential Foxf1-binding sites in the Notch-2 receptor promoter. Nuclear protein extract was prepared from cytomegalovirus (CMV)-Foxf1-transfected or untransfected (UN) U2OS cells and used for EMSA with oligonucleotides containing the high-affinity Foxf1-binding site HFH1 oligonucleotide 25 (44, 48) or potential Foxf1-binding sites in the Notch-2 receptor promoter region (-984/-1,006 and -2,813/-2,786). Specificity of these Foxf1 protein-DNA complexes was demonstrated by the ability of Foxf1 antibody (Ab) and cold self-competition (C, 500-fold molar excess) to interfere with formation of Foxf1 protein-DNA complexes. Formation of the Foxf1 protein-DNA complex was not observed with untransfected U2OS nuclear extracts.

 

Significant reductions in pulmonary expression of the Notch-2 receptor continued in newborn low-Foxf1(+/-) mice (Fig. 4A) and were associated with pulmonary hemorrhage and defects in the peripheral lung microvasculature (25). In newborn high-Foxf1(+/-) lungs, Notch-2 receptor and ADAM-10 mRNA levels and expression of Hes-1 protein were increased to levels found in newborn WT lungs (Fig. 4, A and B), suggesting that Notch-2 signaling was completely restored in newborn high-Foxf1(+/-) lungs. Consistent with this finding, similar protein expression patterns of the Notch-2 receptor were found in newborn WT and high-Foxf1(+/-) lungs (Fig. 3, K and L). Because high-Foxf1(+/-) lungs displayed compensatory increases in Foxf1 mRNA, our results suggest that elevated Foxf1 levels were associated with restored expression of genes in the Notch-2 signaling pathway, correlating with proper development of the peripheral lung microvasculature (25).

Normal proliferation in embryonic Foxf1(+/-) lungs is associated with diminished expression of the negative cell cycle regulators Rb protein, p107, p53, and p21Cip1. Our analysis of Foxf1(+/-) lungs displayed reduced Notch-2 signaling, as evidenced by diminished expression of the Notch-2 receptor and Hes-1 as well as decreased levels of the HGF receptor c-Met (Table 1, Figs. 1 and 2). Activation of Notch-2 signaling is required to maintain proliferation of undifferentiated cells during development (57), and HGF signaling stimulates lung regeneration (42). We therefore examined cellular proliferation in 18-day pc embryonic Foxf1(+/-) lungs using antibodies specific to proliferating cell nuclear antigen or measured DNA replication in newborn lungs by immunohistochemical staining of BrdU incorporation. Surprisingly, these studies demonstrated that proliferation was normal in embryonic Foxf1(+/-) lungs and newborn high-Foxf1(+/-) lungs (data not shown). These results suggest that Foxf1(+/-) lungs possess cellular mechanisms that stimulate proliferation and thus compensate for diminished Notch-2 and HGF signaling. Consistent with these findings, embryonic Foxf1(+/-) lungs exhibited decreased expression of Rb and the Rb-related p107 tumor suppressors (Table 1), and these proteins prevent cell cycle progression by forming inhibitory complexes with the proliferation-specific E2F transcription factor (15, 18, 39). Western blot analysis confirmed significant decreases in Rb protein levels and a 60% reduction in tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21Cip1 protein, in embryonic Foxf1(+/-) lungs (Fig. 5A). In contrast, protein levels of p21Cip1 and Rb protein and GR mRNA expression were normal in newborn and adult high-Foxf1(+/-) lungs compared with age-matched WT lungs (Fig. 5, B and C). Although GR and Rb protein play a critical role in maturation of the lung (4, 29), the significance of GR and Rb protein expression levels to the Foxf1(+/-) lung phenotype remains unclear.



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  		Fig. 5. Embryonic Foxf1(+/-) lungs exhibit reduced levels of cell cycle inhibitor retinoblastoma (Rb), p53, and p21Cip1 proteins and GR, but their levels were restored in newborn high-Foxf1(+/-) lungs. Total protein extracts isolated from 18-dpc (A), newborn (B), and adult (C) Foxf1(+/-) or WT lungs were subjected to Western blot analysis using antibodies against Rb, p21Cip1, p53, and {beta}-actin (loading control). Protein levels were normalized to {beta}-actin. Values are means ± SD. D: increased GR expression in high-Foxf1(+/-) newborn mice. Total lung RNA was isolated from newborn WT and high-Foxf1(+/-) mice and used for RPA with GR and ribosomal protein L32 probe. Expression levels were calculated as described in Fig. 4 legend.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies have shown that haploinsufficiency of the Foxf1 transcription factor caused defects in mesenchymal-epithelial cell signaling, leading to a variety of developmental defects of the lung (25, 31, 34). Half of the Foxf1(+/-) newborn mice, which have only 80% of pulmonary WT levels of Foxf1 mRNA [low Foxf1(+/-) mice], died from pulmonary hemorrhage and exhibited severe defects in development of peripheral lung saccules and microvasculature and severe fusions of the right lung lobes (25, 31, 34). Interestingly, 40% of the surviving newborn Foxf1(+/-) mice displaying compensatory increases in pulmonary levels of Foxf1 mRNA [high-Foxf1(+/-) mice] exhibited normal development of peripheral lung microvasculature (25). In the present study, cDNA probes from 18-day pc embryonic Foxf1(+/-) lungs were used for analysis of Affymetrix microarrays to identify pulmonary genes in which altered expression is associated with developmental abnormalities in low-Foxf1(+/-) lungs (Table 1). The Notch-2-signaling pathway was diminished in embryonic Foxf1(+/-) lungs, as evidenced by significantly reduced levels of the Notch-2 receptor and the Notch-2 downstream target, the transcriptional repressor Hes-1, but a more severe reduction in Notch-2 signaling was found in embryonic low-Foxf1(+/-) lungs, correlating with lower levels of Foxf1 mRNA.

Continued disruption of pulmonary Notch-2 signaling in newborn low-Foxf1(+/-) mice was associated with defects in microvascular development, resulting in lung hemorrhage, and the mice failed to compensate for Foxf1 haploinsufficiency (25). Increased apoptosis of endothelial and smooth muscle cells was observed in newborn low-Foxf1(+/-) lungs (25), a finding consistent with the critical role of Notch-2 signaling in cell survival during mouse embryonic development (14). Interestingly, we show that newborn high-Foxf1(+/-) lungs exhibited normal development of the peripheral lung microvasculature, and Notch-2 signaling was increased to levels found in newborn WT lungs, correlating with compensatory increases in Foxf1 mRNA. Moreover, we show that the Notch-2 receptor is expressed in the lung mesenchyme during mouse embryonic development, suggesting that Foxf1 and Notch-2 possess overlapping expression patterns. In addition to these findings, Foxf1 directly binds to -984/-1006 and -2813/-2786 regions of the mouse Notch-2 receptor promoter, supporting the hypothesis that the Notch-2 receptor is a direct target for Foxf1 transcriptional regulation. These studies indicated that Foxf1 haploinsufficiency caused disruption of Notch-2 signaling and that restoration of the Notch-2 signaling pathway in newborn high-Foxf1(+/-) lungs was associated with increased Foxf1 levels and normal microvascular development and survival.

Recent studies demonstrated that Foxf1 is expressed in hepatic fibroblasts called stellate cells during liver development and in the adult liver (24). In response to CCl4 liver injury, Foxf1(+/-) mice exhibited defective activation of stellate cells, which failed to differentiate into myofibroblasts, resulting in severe hepatocyte apoptosis (24). Interestingly, defective stellate cell activation and increased hepatocyte apoptosis were associated with impaired induction of Notch-2 receptor expression, suggesting that Foxf1 may also regulate Notch-2 signaling in stellate cells during liver regeneration (24).

Activation of Notch-2 signaling is required for maintenance of undifferentiated cell proliferation during development (57). To compensate for diminished proliferation-specific signaling, we speculate that embryonic Foxf1(+/-) lungs exhibited transient reductions in levels of the cell cycle inhibitor Rb and Rb-related p107, and p53 and p21Cip1 proteins, which may play a role in stimulating proliferation in embryonic Foxf1(+/-) lungs. Interestingly, newborn high-Foxf1(+/-) lungs exhibited increased protein levels of Rb and p21Cip1 and increased expression of GR mRNA and correlated with restoration of the Notch-2-signaling pathway. Although GR and Rb play a critical role in maturation of the lung (4, 29), the significance of GR and Rb expression levels to the Foxf1(+/-) lung phenotype remains unclear.

Interestingly, {alpha}1-collagen type XVIII expression was selectively increased in low-Foxf1(+/-) lungs, which display a perinatal lung hemorrhage phenotype (25). The {alpha}1-collagen type XVIII protein is deposited in the basement membrane between epithelial and endothelial cells (52) and contains a protein region homologous to endostatin, which is capable of inhibiting endothelial cell proliferation, angiogenesis, and tumor growth (43). Its increased expression in low-Foxf1(+/-) lungs may be compensating for disruption of the basement membrane interface between type I epithelial and endothelial cells (25). Decreased expression of transcription factors BMI-1, ATF-2, SP-3, and GR mRNA was observed in Foxf1(+/-) lungs, suggesting that Foxf1 haploinsufficiency was associated with disruption of transcriptional pathways. Targeted deletion of the SP-3 (5), GR (4), or ATF-2 (33) gene caused defects in lung formation and maturation, resulting in respiratory failure, supporting the possibility that diminished expression of these genes contributes to embryonic defects in Foxf1(+/-) lung. Consistent with the decreased expression of the GR in embryonic Foxf1(+/-) lungs, these lungs exhibited diminished expression of branched-chain {alpha}-ketoacid dehydrogenase E2, a known target gene for GR activation (12). Furthermore, a 70% reduction in expression of the tight junction protein ZO-1 mRNA was observed and is consistent with disruption of the epithelial-endothelial interfaces found in newborn low-Foxf1(+/-) lungs (25). Interestingly, low-Foxf1(+/-) lungs displayed a 50% reduction in surfactant protein B levels (25), suggesting that Foxf1 haploinsufficiency may affect development of pulmonary epithelial cells. Reduced levels of myosin VI mRNA, which is likely to diminish clathrin-mediated endocytosis (7), were observed in Foxf1(+/-) lungs. These findings suggest that Foxf1 haploinsufficiency is associated with reduced expression of genes that are essential for proper lung development. An alternative explanation for their decreased expression may result from a reduction in the number of various cell types, which was caused by Foxf1 haploinsufficiency.

In summary, Foxf1(+/-) lung defects are associated with diminished expression of genes involved in cell cycle regulation and the Notch-2- and HGF-signaling pathways and several transcription factors involved in normal maturation and development of the lung (ATF-2, BMI-1, SP-3, and GR). Embryonic Foxf1(+/-) lungs exhibited diminished expression of genes in the Notch-2-signaling pathway, the severity of which correlated with pulmonary Foxf1 mRNA levels. Disruption of Notch-2 signaling in newborn low-Foxf1(+/-) lungs was associated with the lung hemorrhage phenotype, whereas Notch-2 signaling and normal microvascular development were restored in high-Foxf1(+/-) lungs. These results suggest that the Foxf1 transcription factor regulates a variety of pulmonary genes required for proper lung morphogenesis and development of the pulmonary microvasculature.


   ACKNOWLEDGMENTS
 
We thank P. Raychaudhuri, X. Wang, K. Krupczak-Hollis, Y. Zhou, M. Major, and F. Rausa for critically reviewing the manuscript.

GRANTS

This work was supported by American Heart Association Scientist Development Grant 0335036N (V. V. Kalinichenko) and National Institutes of Health Grants HL-62446 and AG-21842 (R. H. Costa) and HL-56387 and HL-41496 (J. A. Whitsett).


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. V. Kalinichenko and R. H. Costa, Dept. of Biochemistry and Molecular Genetics (M/C 669), College of Medicine, Univ. of Illinois at Chicago, 900 S. Ashland Ave., Rm. 2220 MBRB, Chicago, IL 60607-7170 (E-mail: vkalin{at}uic.edu and robcosta{at}uic.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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