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First published online 11 April 2007
doi: 10.1242/dev.02846

Development 134, 1991-2000 (2007)
Published by The Company of Biologists 2007
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Foxp2 and Foxp1 cooperatively regulate lung and esophagus development

Weiguo Shu1, Min Min Lu1, Yuzhen Zhang1, Philip W. Tucker2, Deying Zhou1 and Edward E. Morrisey1,3,*

1 Cardiovascular Institute, University of Pennsylvania, 956 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104, USA.
2 Department of Molecular Genetics and the Institute for Cellular and Molecular Biology, University of Texas at Austin, TX, USA.
3 Department of Cell and Developmental Biology, University of Pennsylvania, 956 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104, USA.


Figure 1
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  		Fig. 1. Expression of Foxp1 and Foxp2 during lung and esophagus development. Previously characterized rabbit polyclonal antibodies were used to perform immunohistochemistry on E14.5 embryonic and adult mouse lung tissue sections to determine the expression pattern of Foxp1 and Foxp2. (A,B) Expression is observed for both proteins in the distal tips of the developing airway epithelium at E14.5 (arrowheads). Foxp1 is expressed at low levels in developing bronchial airways, whereas Foxp2 is not (asterisk). (C,D) In adult lungs, Foxp1 is expressed primarily in AEC-2 cells (C, red arrowheads), whereas Foxp2 is expressed in both AEC-2 (D, red arrowheads) and AEC-1 (D, green arrowheads) cells. (E,F) Foxp1 and Foxp2 are both expressed in the muscular component of the developing esophagus at E14.5, whereas only Foxp1 is expressed in the epithelium. epi, epithelium; mu, muscular layers. Scale bars: 100 µm in A,B; 50 µm in C,D; 10 µm in E,F.

 

Figure 2
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  		Fig. 2. Loss of Foxp2 leads to postnatal lung alveolarization defects. (A-F) Hematoxylin and Eosin staining of wild-type and Foxp2-/- mutant mice revealing dilated airspaces in the Foxp2 mutant lungs at E18.5, P8 and P20. (G) Mean linear intercept (MLI) calculation of wild-type and Foxp2 mutant lungs at the indicated times showing airspace enlargement in Foxp2 mutant mice. (H,I) TEM of wild-type (H) and Foxp2 mutant lungs (I) does not reveal a significant change in alveolar epithelial morphology at P8. Scale bars: 500 µm in A-F.

 

Figure 3
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  		Fig. 3. T1alpha is a direct in vivo target of Foxp2 and Foxp1 in the lung. (A-H) Immunohistochemical detection of the expression of SP-C (A,B), T1alpha (C,D), SP-B (E,F) and CC10 (G,H) in wild-type (A,C,E,G) and Foxp2 mutant (B,D,F,H) mouse lungs at E18.5, showing elevated T1alpha expression in the Foxp2 mutant. Arrows, AEC-2 cells; arrowheads, lymphatic vessel. (I-K) Co-staining for expression of Foxp2 (I) and T1alpha (J) reveals that both proteins are co-expressed in the same cells within the distal lung at E18.5 (K). Note that the dim fluorescence displayed by cells located between the airways in I is non-specific background. (L,M) Expression of T1alpha in lymphatic endothelium at E18.5 (arrows) does not differ between wild-type and Foxp2-/- lungs in contrast to the upregulation of T1alpha expression in alveoli (asterisk) of the mutant. (N) Q-PCR to assess expression of the genes encoding the indicated lung cell markers at E18.5 confirming specific upregulation of T1alpha (T1{alpha}) expression in Foxp2-/- lungs. Values are relative to wild-type expression levels, which are set at 1.0 for each gene. aqua-5, aquaporin 5. Q-PCR results are the average of three lung samples performed in triplicate±s.e.m.; *, P<0.001. (O) Luciferase reporter assays were performed in NIH-3T3 cells using the rat 1.3 kb T1alpha (T1{alpha}) promoter. Fox DNA-binding sites located in the T1alpha promoter are indicated by either green ovals (conserved between mouse and rat) or white ovals (present in the rat promoter but not conserved across species). Comparison between the rat sequence (upper sequence) and the mouse sequence (lower sequence) in the two conserved sites in the T1alpha promoter. Bar chart showing that co-expression of either Foxp1 or Foxp2 leads to a fivefold repression of the T1alpha promoter relative to pCMV. (P) ChIP assays performed using mouse lung chromatin and the previously characterized Foxp1 and Foxp2 polyclonal antibodies show that Foxp1 and Foxp2 are both found associated with the region containing the conserved Fox DNA-binding sites in the mouse T1alpha promoter in vivo. Scale bars: 50 µm in A-M.

 

Figure 4
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  		Fig. 4. Foxp2-/-;Foxp1+/- compound mutants exhibit dramatic defects in lung airway morphogenesis. Foxp2-/-;Foxp1+/- compound mutants were generated by crossing Foxp1+/-;Foxp2+/- double heterozygous mice to Foxp1+/- mice. (A-D) Overall lung size was reduced at both E14.5 and E18.5 in Foxp2-/-;Foxp1+/- mutants. (E-J) At E14.5, E16.5 and E18.5, significant defects were observed in airway development including decreased branching morphogenesis as demonstrated by the dilated nature of the developing airways. (K) Distal airspace area, as measured using ImageJ software, was significantly increased in Foxp2-/-;Foxp1+/- lungs at both E14.5 and E18.5. (L-Q) Despite these defects, proximal-distal epithelial patterning was maintained in Foxp2-/-;Foxp1+/- compound mutants as determined by normal expression patterns of SP-C (L,O), CC10 (M,P) and ß-tubulin IV (N,Q) proteins. Scale bars: 500 µm in A-J,L,M,O,P; 100 µm in N,Q.

 

Figure 5
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  		Fig. 5. Decreased expression of N-myc and Hop in Foxp2-/-;Foxp1+/- compound mutant mice. In situ hybridization was performed on wild-type (A,C,E,G,I,K,M,O) and Foxp2-/-;Foxp1+/- compound mutants (B,D,F,H,J,L,N,P) at E16.5 to determine the lung epithelial expression of genes encoding SP-C (A,B), Nkx2.1 (C,D), Shh (E,F), SP-B (G,H), Foxa2 (I,J), Gata6 (K,L), N-myc (M,N), and Hop (O,P). (Q) Expression of N-myc and Hop was significantly decreased in Foxp2-/-;Foxp1+/- compound mutants, whereas expression of Nkx2.1 was not significantly affected as measured by Q-PCR. Scale bars: 100 µm in A,B; 500 µm in C-P.

 

Figure 6
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  		Fig. 6. Decreased epithelial and mesenchymal cell proliferation in Foxp2-/-;Foxp1+/- compound mutant lungs. Immunohistochemistry was performed using a Ki-67 antibody to detect proliferating cells in the lungs of wild-type (A) or Foxp2-/-;Foxp1+/- compound mutant (B) mice. Quantitation (C) shows that there is a more than 40% reduction in cell proliferation in both the epithelia and mesenchyme of Foxp2-/-;Foxp1+/- compound mutant lungs. Expression of cyclin D1 (D,E) is decreased in Foxp2-/-;Foxp1+/- lungs, whereas expression of the CDKI p57 (F,G) is increased. Scale bars: 50 µm.

 

Figure 7
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  		Fig. 7. Foxp2-/-;Foxp1+/- mutants have defects in esophageal muscle development. (A-F) Esophageal development was examined by Hematoxylin and Eosin staining in wild-type (A,B), Foxp2-/- mutant (C,D) and Foxp2-/-;Foxp1+/- mutant (E,F) mice at E18.5. As early as E14.5, the smooth muscle surrounding the esophagus was thinner in Foxp2-/-;Foxp1+/- mutants than in either Foxp2-/- or wild-type littermates (A,C,E). By E18.5, Foxp2-/-;Foxp1+/- mutants had severely dilated esophagi with a very thin muscular layer (B,D,F). (G-J) Smooth muscle actin (sm {alpha}-actin) staining revealed a single muscular layer surrounding Foxp2-/-;Foxp1+/- esophagi as compared with wild-type animals (black arrowheads). This single muscular layer also appeared thicker in Foxp2-/-;Foxp1+/- mutants (I,J). The sm-actin-positive submucosal layer was unchanged in Foxp2-/-;Foxp1+/- mutants (white arrowheads). (K,L) MyoD immunohistochemistry demonstrated the presence of skeletal muscle in the esophagi of wild-type embryos (K, arrowheads) and revealed a lack of skeletal muscle contribution to Foxp2-/-;Foxp1+/- esophagi (L). Scale bars: 100 µm in A-F; 50 µm in G-L.

 

Figure 8
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  		Fig. 8. Apoptosis and cell proliferation in the esophagus of Foxp2-/-;Foxp1+/- mutant mice. TUNEL staining (A,B) and Ki-67 immunostaining (C,D) were performed on wild-type (A,C) and Foxp2-/-;Foxp1+/- (B,D) esophagi at E14.5. No difference in either apoptosis or cell proliferation was detectable.

 




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