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Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme

Department of Molecular Biology and Pharmacology, Washington University Medical School, Campus Box 8103, 660 S. Euclid Avenue, St Louis, MO 63110, USA

View larger version (95K): [in a new window]   Fig. 1. Morphological and histological analysis of Fgf9-/- lungs. (A,B) Anterior views of gross dissections of uninflated control (A) and Fgf9-/- (B) lungs at E18.5. The Fgf9-/- lung is smaller but contains the same lobes as the control. Arrowhead in B indicates the accessory lobe. (C,D) Sagittal sections of the right side of the thoracic cavity at similar levels from E14.5 control (C) and Fgf9-/- (D) embryos. The control lung (lu) fills the thoracic cavity while the Fgf9-/- lung does not. A dilated cardiac atrium (a) is observed adjacent to the Fgf9-/- lung (D). An intact diaphragm (arrowheads) is seen just superior to the liver (li) in both C,D. (E,F) High power views of the caudal part of the right lower lobe of grossly dissected E12.5 control (E) and Fgf9-/- (F) lungs. Note the lack of mesenchyme and the reduced branching complexity in the Fgf9-/- lung relative to the control. (G,H) High-power views of the left lobe of grossly dissected E12.5 control (G) and Fgf9-/- (H) lungs. Epithelial buds in the Fgf9-/- lung (arrow in H) appear dilated and have less adjacent mesenchyme than comparably located epithelial buds in the control lung (arrow in G). In addition, a control lung bud (arrow in G) is branching into two buds, while the corresponding Fgf9-/- bud (arrow in H) is not branching. (I,J) Sagittal sections of E11.5 lungs showing similar appearance of control (I) and Fgf9-/- (J) samples. (K,L) E13.5 lung sections showing reduced mesenchyme in Fgf9-/- (L) compared with control lung (K). Small diameter airways are more prevalent in control lung (arrow in K) than in Fgf9-/- lung. (I-L) are shown at the same magnification. m, mesenchyme; r, ribs; asterisks indicate future airspaces within epithelial branches.

View larger version (88K): [in a new window]   Fig. 2. Analysis of airway branching in E18.5 Fgf9-/- lungs using epithelial casts. (A-C) Epithelial casts of whole control (A) and Fgf9-/- (C) lungs. The Fgf9-/- lung cast is smaller than the control but exhibits evidence of both proximal airway branching and distal airspace formation. Some proximal branches are observed in the upper part of the Fgf9-/- cast, and alveolar sacs covering the surface indicate generation of distal airways. (B) An underinjected epithelial cast of a control lung showing a network of proximal branches that is larger than the total size of the Fgf9-/- cast (C). (D,E) Accessory lobe casts dissected from whole lung epithelial casts of control (D) and Fgf9-/- (E) lungs. (E is shown at 1.2x the magnification of D.) The lung cast shown in (E) was overinjected to ensure filling of all airway branches. Note the absence of branches along much of the Fgf9-/- accessory lobe bronchus (arrow), while further iterations of branches obscure the main bronchus of the control accessory lobe. (F,G) High-power views of the dorsal side of the left lobe of control (F) and Fgf9-/- (G) lung casts showing a similar density of alveolar sacs on the lung surface. (H,I) Scanning electron microscope images of control (H) and Fgf9-/- (I) lung casts, showing similar structure of distal clusters of alveolar sacs. The normal structure of dense tufts of alveolar sacs in (I), and the normal density of surface alveolar sacs in (G), together suggest that significant distal airspace formation occurs in Fgf9-/- lung. (J) Schematic diagram of the progression of epithelial airways from proximal conducting airways to distal respiratory airways in embryonic lung. Respiratory airways are distinguished from conducting airways by the presence of alveolar sacs in the walls of respiratory airways. (Respiratory bronchioles (rb), which serve as both conducting and respiratory airways in humans and in many animals, are not found in rodents.) Alveoli are formed by septation of alveolar sacs, a process that occurs in mice only after birth. Scale bars: 50 µm in H,I. ad, alveolar duct; as, alveolar sac; br, bronchus; rb, respiratory bronchiole; tb, terminal bronchiole; tr, trachea.

View larger version (137K): [in a new window]   Fig. 3. Normal distal and proximal differentiation in E18.5 Fgf9-/- lungs. (A-D) Toluidine Blue stained plastic sections of control (A,C) and Fgf9-/- (B,D) lungs showing normal differentiation of distal (A,B) and proximal (C,D) epithelium. (A,B) Sections of control (A) and Fgf9-/- (B) distal lung showing similar epithelial structure between alveolar sac airspaces. Surfactant (s) is seen in airways in A,B. Note that capillaries (white arrows) are present and similarly located within surrounding tissue in A,B. Differentiating epithelial cells display characteristic vacuolization due to loss of glycogen during section processing (black arrows in A,B). (C,D) Sections of proximal airway in control (C) and Fgf9-/- (D) lungs show similar epithelial architecture. Normal appearing ciliated cells (arrowheads) and Clara cells (arrows) are seen in control (C) and Fgf9-/- (D) sections. (E-L) Transmission electron micrographs of E18.5 control and Fgf9-/- lungs. (E,F) Control (E) and Fgf9-/- (F) lung capillaries showing the gas diffusion barrier between airspace (a) and blood (b). (G,H) Higher power views of the diffusion barrier in control (G) and Fgf9-/- (H) lungs, demonstrating normal close apposition of endothelial cell processes (e) and Type I pneumocyte processes (p), separated by basement membrane (asterisks). (I) A Type II pneumocyte from a control lung showing lamellar bodies (asterisk). (J) A Type II pneumocyte from an Fgf9-/- lung showing lamellar bodies (asterisk) and surface microvilli (arrowhead). (K) Transmission electron microscopy of the surface of an Fgf9-/- lung showing the basement membrane (arrowheads) beneath and a long microvillus (arrow) on the surface of a pleural mesothelial cell. (L) Transmission electron micrograph of the surface of an Fgf9-/- lung showing a tight junction (arrowheads) between two pleural mesothelial cells. (The tight junction is the extended s-shaped region of close apposition between the plasma membranes of neighboring pleural cells.) Scale bars: 1.1 µm in E,F; 0.15 µm in G,H; 0.5 µm in I,J; 0.28 µm in K; 0.22-µm in L.

View larger version (134K): [in a new window]   Fig. 4. Immunohistochemistry for epithelial and vascular markers in E18.5 Fgf9-/- lungs. (A,B) Hematoxylin and Eosin stained sections of the left lobe of control (A) and Fgf9-/- (B) lungs. Note that fewer generations of conducting airways (i.e. airways without alveolar sacs in their walls) are seen in the Fgf9-/- lungs compared with controls. (The arrow in A indicates a conducting airway.) Distal aspects of both lungs have similar alveolar duct and alveolar sac structures, which are particularly evident in the less expanded upper regions of the lung sections (leftmost regions of (A,B)). (C,D) Immunohistochemistry for CCSP in lung sections from control (C) and Fgf9-/- (D) embryos. Fgf9-/- lung (D) shows normal expression of CCSP in Clara cells in proximal airways. Note the relatively larger diameter of CCSP-positive airways (arrowheads) in Fgf9-/- versus control lung, indicating reduced branching of conducting airways. (E,F) Immunohistochemistry for SP-C in lung sections from control (E) and Fgf9-/- (F) embryos. Fgf9-/- lung (F) shows expression of surfactant in Type II pneumocyte cytoplasm that is comparable with that in the control (E). (G,H) Immunohistochemistry for vWF in lung sections from control (G) and Fgf9-/- (H) embryos, showing normal staining of large vessels in Fgf9-/- lung.

View larger version (60K): [in a new window]   Fig. 5. Whole-mount in situ hybridization for Shh and Fgf10 in embryonic Fgf9-/- lungs. (A,B) In situ hybridization for Shh in E14.5 control (A) and Fgf9-/- (B) lungs. Epithelial Shh expression in Fgf9-/- lung is comparable with that in control lungs. (C-F) Fgf10 expression in E12.5 control (C,E) and Fgf9-/- (D,F) lungs. (E,F) Higher magnification images of the boxed regions in C,D, respectively. Note comparable expression of Fgf10 in mesenchyme distal to airway buds in control (C,E) and Fgf9-/- (D,F) lungs. (G-J) Fgf10 expression in E14.5 control (G,I) and Fgf9-/- (H,J) lungs. Fgf9-/- lung (H) shows reduced expression of Fgf10 relative to control lung (G). Note the absence of Fgf10 expression in the region of H indicated by the arrow and the presence of Fgf10 expression in the corresponding region of G. The indicated Fgf10 negative buds (arrow in H) are dilated with little surrounding mesenchyme (similar to buds in Fig. 2J); this suggests that absence of Fgf10 expression may be due to a lack of mesenchymal tissue. (H is shown at 1.4x the magnification of G.) (I,J) Higher magnification images of the boxed regions of G,H, respectively. Note control Fgf10 expression in the mesenchyme between epithelial buds (arrowheads in I). No Fgf10 expression is seen in the comparable regions of the Fgf9-/- lung (arrowheads in J). (I,J are at the same magnification.)

View larger version (51K): [in a new window]   Fig. 6. Models for molecular signaling in developing lung. (A) Reciprocal FGF signaling between epithelium and mesenchyme during lung (top) and limb (bottom) embryogenesis. (A, top) We propose that FGF9 from the lung epithelium and pleura activates FGFR1c in the mesenchyme, while FGF10 from the mesenchyme activates FGFR2b in the epithelium. (A, bottom) Similar essential reciprocal epithelial-mesenchymal signaling between FGF10 in the mesenchyme and FGF8 in the overlying apical ectodermal ridge (AER) regulates limb bud outgrowth in mouse embryos. (DM, distal mesenchyme; ZPA, zone of polarizing activity). (FGF8 and FGF9 may also signal through FGFR2c, but Fgfr1 is more highly expressed than Fgfr2 in embryonic lung and limb mesenchyme (Peters et al., 1992).) (B) Model for interactions between Fgf and non-Fgf signaling pathways in developing lung. (B, top) Signaling during proximal airway branching in the early pseudoglandular period. (1) FGF9 (blue) in both airway epithelium and pleura stimulates mesenchymal proliferation. (Grey shading indicates mesenchyme.) (2) FGF10 (red) in the mesenchyme induces airway branching by stimulating endoderm proliferation and migration. (3) SHH (hatched) in the airway epithelium promotes mesenchymal proliferation. (B, middle) Signaling during airway branching in the late pseudoglandular period. (1) FGF9, now limited to the pleura, stimulates mesenchymal proliferation. (2) Mesenchymal FGF10 stimulates endoderm proliferation and migration, and appears to induce distal epithelial BMP4 expression (yellow). (3) BMP4 appears to inhibit airway branching by inhibiting endoderm proliferation, and perhaps by inhibiting endoderm migration. (4) SHH in the epithelium stimulates mesenchymal proliferation. Vascular defects in Shh-/- lungs indicate that Shh regulates development of mesenchymal vasculature. SHH also appears to prevent more generalized mesenchymal expression of Fgf10 (truncated symbol). (B, bottom) Signaling during development of distal airspaces. Mesenchyme surrounding epithelial buds is sharply reduced; focally high Fgf10 expression in the adjacent mesenchyme is lost; and epithelial differentiation begins with the narrowing of the epithelium. (1) Epithelial Bmp4 expression is essential for distal epithelial differentiation. (2) Continued Shh expression in the epithelium may regulate vascular development. In this model, distal lung development proceeds when mesenchyme surrounding developing airways thins, Fgf10 expression is reduced and Bmp4 expression is high. In Fgf9-/- lungs, loss of Fgf9-induced mesenchymal proliferation could result in premature reduction in mesenchyme surrounding budding airways, leading to premature reduction in Fgf10 expression. This would allow distal lung development to commence after fewer iterations of airway branching.

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