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First published online February 2, 2004
doi: 10.1242/10.1242/dev.00966

Development 131, 953-964 (2004)
Published by The Company of Biologists 2004
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Foxa2 regulates alveolarization and goblet cell hyperplasia

Huajing Wan1, Klaus H. Kaestner2, Siew-Lan Ang3, Machiko Ikegami1, Fred D. Finkelman4,5, Mildred T. Stahlman6, Patricia C. Fulkerson7, Marc E. Rothenberg7 and Jeffrey A. Whitsett1,*

1 Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
2 Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6145, USA
3 Division of Developmental Neurobiology, National Institute for Medical Research, London NW7 1AA, UK
4 Cincinnati Veterans Administration Medical Center, 3200 Vine Street, Cincinnati, OH 45220, USA
5 Division of Immunology, University of Cincinnati College of Medicine, 231 Sabin Way, Cincinnati, OH 45267, USA
6 Department of Pediatrics, Vanderbilt University, Nashville, TN 37232-2370, USA
7 Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA



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  		Fig. 1. (A) Conditional control of Foxa2 gene targeting. In triple transgenic mice (SP-C-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP and CCSP-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP), rtTA is expressed in epithelial cells under the control of either the human SP-C or CCSP promoters. In the presence of doxycycline, rtTA binds to the (tetO)7CMV promoter and activates the expression of Cre-recombinase, causing recombination and deletion of exon 3 in Foxa2loxP/loxP mice, and producing Foxa2{Delta}/{Delta} mice. (B) Immunohistochemistry demonstrates Foxa2 deletion. Lung sections were prepared on PN16 for immunohistochemistry using anti-FOXA2 antibody. Triple transgenic mice, SP-C-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP (B part B) and CCSP-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP (B part D), and littermate control mice (B part A, B part C) were maintained on doxycycline from E0. Inserts are higher magnifications (x40) of the regions indicated by arrowheads. Nuclear FOXA2 staining was observed in epithelial cells of conducting and peripheral airways and alveoli, and was absent or decreased in Foxa2{Delta}/{Delta} mice. Goblet cell hyperplasia was observed in both SPC-rtTA and CCSP-rtTA Foxa2{Delta}/{Delta} mice (insets). Figures are representative of at least four individual mice. Scale bar, 50 µm. (C) RNA protection assay for estimation of Foxa2 mRNA. RNA protection assays were used to quantitate Foxa2 mRNA in lungs from SP-C-rtTA, Foxa2{Delta}/{Delta} and control littermates at E18.5 and compared with L32 mRNA. Dams were treated with doxycycline from E0 to E18.5.

 


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  		Fig. 2. Effects of Foxa2-deletion on lung morphogenesis. Triple transgenic mice (SP-C-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP) and littermate controls were maintained on doxycycline from E0. (A-F) Lung sections of triple transgenic mice and littermate controls were prepared on E18.5 (A,B), PN3 (C,D) and PN16 (E,F) and stained with Hematoxylin and Eosin to assess lung morphology. Figures are representative of at least four individual mice. Scale bar: 300 µm.

 


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  		Fig. 3. Morphometric analysis. Fractional airspace and fractional lung parenchyma were calculated in lungs of mice from each genotype after exposure to doxycycline for the defined times. Fractional airspace ratio was significantly increased in SP-C-rtTA but not CCSP-rtTA, Fox2{Delta}/{Delta} mice. *P<0.05 vs control mice (Student's t-test).

 


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  		Fig. 4. Goblet cell hyperplasia after Foxa2 deletion. (A-H) Compound transgenic mice, either SP-C-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP (C,E,G) or CCSP-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP (D,F,H), and controls (non-Foxa2 deleted) (A,B) were treated with doxycycline from E0. At PN16, lung sections were stained for FOXA2 (arrows) and either Alcian Blue or periodic acid Schiff (PAS). Staining by Alcian Blue was observed in cells with goblet cell morphology (A-D, arrowheads). Similarly, PAS (E,F, arrowheads) and MUC5AC staining (G,H, arrowheads) were observed in goblet cells in conducting airways in Foxa2-deleted mice. FOXA2 staining was absent in nuclei of the goblet cells (C-H). Figures are representative of at least four individual mice of each genotype. Scale bar: 200 µm.

 


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  		Fig. 5. Timed deletion of Foxa2 during lung morphogenesis. The period of doxycycline treatment is shown in the top panel. Lung sections from SP-C-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP (B,D,F,H) and littermate controls (A,C,E,G) were prepared on PN16 and stained with hematoxylineosin. Airspace enlargement and neutrophilic infiltrations were observed in the Foxa2-deleted mice. Figures are representative of at least four individual mice of each genotype. Scale bar: 300 µm.

 


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  		Fig. 6. FOXA1, FOXJ1, PECAM and elastin staining. Lung sections were prepared on PN16. Triple transgenic mice, SP-C-rtTA-/tg, (tetO)7CMV-Cre-/tg, Foxa2loxP/loxP and littermate controls were maintained on doxycycline from E0. (A-H) Lung sections were stained for FOXA1 (A,B), FOXJ1 (C,D), PECAM (E,F), and elastin (G,H). Inserts are higher magnifications (x40). Figures are representative of at least four individual mice of each genotype. Scale bar: 100 µm.

 


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  		Fig. 7. Pulmonary mechanics (A-F) Airway resistance (A), airway elastance (B), compliance (C), tissue damping (D), tissue elastance (E) and hysteresivity (F) were measured in 7-week-old CCSP-rtTA, Foxa2{Delta}/{Delta} (closed bar, n=6) and control mice (open bar, n=5). Increased airway resistance, airway elastance, tissue damping, tissue elastance and decreased compliance were observed. Values are mean±s.e.m. *P<0.05 vs control mice (Student's t-test).

 


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  		Fig. 8. FOXA2 staining of mouse models with goblet cell hyperplasia. (A-L) Lung sections were prepared from mouse models with goblet cell hyperplasia and stained for FOXA2. Models: overexpression of IL4 (A-C); overexpression of IL13 (D-F); deletion of SP-C (G-I); and ovalbumin challenged mice (J-L). Normal bronchi (A,D,G,J) and bronchi with goblet cell hyperplasia (B,E,H,K) are shown. FOXA2/Alcian Blue staining (C,F,I,L) indicates a close correlation between goblet cell hyperplasia and decreased or absent FOXA2 expression. Figures are representative of at least four individual mice from each model. Scale bars: 50 µm.

 


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  		Fig. 9. Effects of IL4 on FOXA2 are STAT6 dependent. (A-H) Adult, control (A-D) and Stat6-/- (E-H) mice were treated intranasally with IL4. Lung sections were prepared and stained for FOXA2. The mice were treated with either saline (A,B,E,F) or IL4 (C,D,G,H). IL4 induced goblet cell hyperplasia in control but not Stat6-/- mice. Foxa2 staining decreased at sites of goblet cell hyperplasia in wild-type mice. Foxa2 staining and epithelial cell morphology were not perturbed in Stat6-/- mice. Figures are representative of four mice of each genotype. Scale bars: 50 µm.

 


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  		Fig. 10. FOXA2 inhibits Muc5ac transcription. H292 cells were transfected with promoter plasmid pGL3-MUC5AC-luc and increasing amounts of the expression plasmid pRC-CMV-FOXA2. MUC5AC promoter activity was determined by the relative luciferase activity normalized to ß-galactosidase activity. As a positive control, all trans-retinoic acid (3 µgml-1) was added to induce MUC5AC expression. Plasmid pRC-CMV was used as empty vector control. *P<0.05 versus control (ANOVA).

 


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  		Fig. 11. FOXA2 staining of human lung tissue with goblet cell hyperplasia. (A) Photomicrograph (x40) of a small conducting airway of a 5-month-old infant who died with bronchopulmonary dysplasia. The nuclei of Alcian Blue-stained mucus cells are unstained by the FOXA2 antibody. A few cells with FOXA2-stained nuclei are observed in the basal layer of the conducting airways and a nearby terminal airway. (B) Photomicrograph (x10) of the lung from the patient in A, showing a cuboidal lined alveolar duct arising from the small columnar cell lined bronchus. The nuclei of the Alcian Blue-stained mucus cells in the bronchus lack FOXA2, but most of the nuclei of the cells lining the alveolar ducts are FOXA2 reactive. (C) Photomicrograph (x40) of the lung of a 27-year-old female who underwent lobectomy for bronchiectasis. This shows a small bronchus lined with Alcian Blue-stained mucus cells, the nuclei of which lack FOXA2 staining. Many nuclei of non-goblet cells lining terminal airways are FOXA2 reactive. (D). Photomicrograph (x10) of the lung of a 6.5-month-old infant dying with bronchopulmonary dysplasia showing terminal airway cell nuclei immunostained for FOXA2. Nuclei of epithelial cells lining a small conducting airway lack Alcian Blue staining and are immunostained by the FOXA2 antibody. Scale bar: 50 µm.

 

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© The Company of Biologists Ltd 2004