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Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations

¹ Department of Molecular Biology, Göteborg University, The Lundberg Laboratory, Box 462, SE-405 30 Göteborg, Sweden
² Department of Medical Biochemistry, Göteborg University, SE-405 30 Göteborg, Sweden

View larger version (82K): [in a new window]   Fig. 1. Defects in lung lobation and maturation in Foxf1+/- embryos. (A,B) Ventral views of lungs from E18.5 wild-type and Foxf1+/- fetuses. The mutant lungs (A) are small, pale and have hemorrhagic lesions along the edges (arrowhead). The accessory lobe of the right lung is missing and the remaining lobes are partially fused. (C-F) Hematoxylin and Eosin staining of transversal sections from E18.5 Foxf1+/- (C,E) and wild-type (D,F) lungs. Low magnification view (C,D) shows the smaller size and denser appearance of the mutant lung. Higher magnification (E,F) reveals that the lax alveolar structure of the wild-type lung is replaced by compact mesenchyme in the Foxf1 mutant. (G-N) Differentiation of epithelial and mesenchymal cells in E18.5 Foxf1+/- (G,H,K,L) and wild-type (I,J,M,N) lungs. In situ hybridization with probes for Utg (G,I), a marker for Clara cells of the proximal, bronchiolar epithelium, and Sftpc (H,J) expressed in the distal respiratory epithelium. Immunostaining of an endothelial cell marker (K,M; Pecam) and detection of vascular smooth muscle cells by in situ hybridization with a probe for Actvs (L,N). Ac, accessory lobe; Ar, arteriole; Br, bronchiole; Ca, caudal lobe; Cr, cranial lobe; LL, left lung; Me, medial lobe; RL, right lung.

View larger version (132K): [in a new window]   Fig. 2. Foregut, respiratory tract and skeletal malformations in Foxf1+/- embryos. (A-C) Hematoxylin and Eosin stained transversal sections showing trachea (Tr) and esophagus (Es) of E18.5 Foxf1+/- (A,C) and wild-type (B) fetuses. The mutants have variable degrees of narrowing of trachea and esophagus with esophageal atresia and a fistula-like fusion between esophagus and trachea. (D,E) Ventral view of Alcian Blue-stained tracheas from E18.5 Foxf1+/- (D) and wild-type (E) fetuses. The C-shaped cartilaginous rings, which are segmentally arranged along the trachea (E), are replaced by irregular, hypoplastic patches of cartilage in Foxf1+/- embryos (D; compare also B with A,C). (F,G) Skeletal malformation in Foxf1+/- mice shown by Alizarin Red and Alcian Blue staining of E18.5 fetuses. Attachment of the ribs to the sternum is asymmetrical in the mutant, and the ossification centra in the sternebrae (red staining parts of the sternum) are misaligned at the midline (F).

View larger version (77K): [in a new window]   Fig. 3. (A-D) Foxa2 whole-mount in situ hybridization of E11.5 (A,B) and E12.5 (C,D) Foxf1+/- (A,C) and wild-type (B,D) lungs. Primary epithelial branches of the right lung are formed normally at E11.5 in the mutant, although with a tendency of the buds to be wider proximally and less well defined (A). At E12.5, growth of the lungs and epithelial branching are retarded in mutant embryos (C) compared with wild type (D). The mesenchyme surrounding the accessory lobe epithelial bud of the mutant right lung remains fused with that of the caudal lobe (C; arrowhead), while a separate accessory lobe has already formed in wild-type embryos (D). Ac, accessory lobe; Ca, caudal lobe; Cr, cranial lobe; LL, left lung; Me, medial lobe.

View larger version (106K): [in a new window]   Fig. 4. Foxf1 is a target for Sonic hedgehog signaling in lung. (A-C) Gene expression in E11.5 mouse lung examined by whole-mount in situ hybridization. Shh (A) is expressed throughout the lung epithelium, but the highest expression level is restricted to distal epithelium in the bulbous ends of the buds. Ptch (B), encoding the Shh receptor, and Foxf1 (C) are both expressed in mesenchyme adjacent to lung epithelium. The expression patterns mirror the distribution of Shh, with low levels along the proximal tubular epithelium and higher levels distally. (D-G) Exogenous Shh activates Foxf1. COS-7 cells transfected to secrete Shh (D,F) or control COS-7 cells (E,G) were grafted into the mesenchyme (location of grafts indicated by white stars) of wild-type lung explants, which were then cultivated for 24 hours. The expression of Ptch (D,E) and Foxf1 (F,G) were examined by whole-mount in situ hybridization. The mesenchyme immediately adjacent to the Shh-producing cells express elevated levels of both Ptch (D) and Foxf1 (F), whereas no change is induced by control cells (E,G).

View larger version (69K): [in a new window]   Fig. 5. Foregut and sclerotome expression of Foxf1 depends on Shh. Foxf1 whole-mount in situ hybridization of wild-type (A-D) and Shh-/- (E-H) E10.5 (A,B,E,F) or E12.5 (C,G) embryos and E12.5 lungs (D,H). In Shh mutants, Foxf1 expression is missing in the anterior part of the gut, the oral cavity, in sclerotomes and lungs. Foxf1 mRNA persists only in the posterior part of the gut, where Ihh is expressed. Embryos in A,E are in aqueous solution, to visualize the general morphology. Embryos in B,C,F,G are clarified in benzylalcohol:benzylacetate to show staining of internal structures. In, intestine; Li, liver; Lu, lung; OC, oral cavity; Sc, sclerotome.

View larger version (130K): [in a new window]   Fig. 6. Foxf1 expression is downregulated by BMP4 and indirectly by FGF7 and FGF10. Beads soaked in BMP4 (A), FGF10 (C,E) or FGF7 (G,I,K) were implanted in the mesenchyme of wild-type lung explants, which were then cultivated for 24 hours. BSA-soaked beads served as a controls (B,D,F,H,J,L). Expression of Foxf1 (A-D,G,H), Bmp4 (E,F), Ptch (I,J) and Shh (K,L) was analyzed by whole-mount in situ hybridization. BMP4 reduces Foxf1 expression in mesenchyme adjacent to the bead (A). FGF10 attracts distal epithelium and stimulates its proliferation, which is seen as dilation of the bulbous part of buds close to the bead (C). The patches of elevated Foxf1 expression next to distal epithelium (D) are lost in the area influenced by exogenous FGF10 (C), whereas no effect is seen on the low level expression in mesenchyme along the proximal, tubular epithelium (C). A likely explanation for this inhibition is that it is mediated by BMP4, as Bmp4 is activated by FGF10 in distal, but not in proximal epithelium (E). FGF7 induces an even more pronounced epithelial proliferation and bud expansion (G,I,K). Foxf1 is generally downregulated in the area influenced by FGF7 (G), which may be mediated by a decreased Shh signaling, as epithelial Shh expression is reduced by FGF7 (K). This interpretation is supported by the similarity in the effect of FGF7 on Foxf1 and another Shh-responsive gene, Ptch (I).

View larger version (38K): [in a new window]   Fig. 7. Summary of paracrine interactions shaping the Foxf1 expression pattern in developing lung. (A) Activation of Foxf1 by Shh is based on both ectopic expression and loss-of-function experiments presented in this work. The inclusion of Gli2/3 in this pathway is inferred from their role in transducing the hedgehog signal and the similarity between Gli2/3 knockout phenotypes and the Foxf1 heterozygote phenotype. Inhibition by BMP4 refers to the effect of BMP4 beads on Foxf1 expression presented here. The role of FGF10 and FGF7 in regulation of Bmp4 and Shh, respectively, has been published by others (Lebeche et al., 1999) and has also been confirmed by us; the net effect of FGFs on Foxf1 expression was analyzed in this work. (B) High magnification view of lung explant hybridized with a Foxf1 probe. Foxf1 expression in subepithelial mesenchyme peaks at the transition between the proximal, tubular epithelium the distal, bulbous part of the bud. The circular object is a bead implanted in the mesenchyme as a negative control and has no relevance for the expression pattern discussed here. (C) Model of paracrine signaling in a lung bud that provides a possible explanation for the observed distribution of Foxf1 mRNA. Proximal tubular epithelium secretes a low level of Shh (pink), which gives rise to a low level of Foxf1 expression (light blue) in the subepithelial mesenchyme. Shh secretion is higher in the distal epithelium (red), which activates high level expression of Foxf1 (dark blue). Fgf10 is expressed in mesenchyme at the distal tip of the bud (black). In response to FGF10 signaling, Bmp4 is activated in the most distal epithelium (green). Secretion of BMP4 inhibits Foxf1 expression, which therefore drops again towards the tip of the bud (light blue).