. 2009 Jan;238(1):123-37.
Fibroblast Growth Factor 9 Signaling Inhibits Airway Smooth Muscle Differentiation in Mouse Lung
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Fibroblast Growth Factor 9 Signaling Inhibits Airway Smooth Muscle Differentiation in Mouse Lung
2009 Jan .
Free PMC article
In mammalian lungs, airway smooth muscle cells (airway SMCs) are present in the proximal lung adjacent to bronchi and bronchioles, but are absent in the distal lung adjacent to terminal sacs that expand during gas exchange. Evidence suggests that this distribution is essential for the formation of a functional respiratory tree, but the underlying genetic mechanism has not been elucidated. In this study, we test the hypothesis that fibroblast growth factor 9 (Fgf9) signaling is essential to restrict SMC differentiation to the proximal lung. We show that loss of Fgf9 or conditional inactivation of Fgf receptors (Fgfr) 1 and 2 in mouse lung mesenchyme results in ectopic SMCs. Our data support a model where FGF9 maintains a SMC progenitor population by suppressing differentiation and promoting growth. This model also represents our findings on the genetic relationship between FGF9 and sonic hedgehog (SHH) in the establishment of airway SMC pattern.
Copyright (c) 2008 Wiley-Liss, Inc.
Figure 1. Inactivation
Fgfr1;2 in lung mesenchyme results in a reduction in mesenchyme thickness
(A,B) Section of β-gal stained T-Cre;R26R lung at Embryonic day (E) 10.0 shortly after lung initiation. Boxed area in A is magnified in B. Blue-stained mesenchymal and mesothelial cells encase the single layer columnar epithelium that contains a few blue cells. (C,D)
Fgfr1 expression as indicated by RNA in situ hybridization in E10.5 lungs using a probe that hybridizes to the portion of the transcript that is deleted by Cre-mediated recombination (Xu et al., 1999). Fgfr1 expression is detected in the mesenchyme of the control lung (C), but is absent in the Fgfr1;2-DKO lungs (D). (E,F) Transverse sections of E12.5 embryos showing that the chest cavity of the Fgfr1;2-DKO mutant is similar in size compared to control. Arrowheads indicate lungs. (G–J) Brightfield images of lungs. Boxed regions are magnified in the insets. Red lines indicate the mesenchymal thickness at the caudal tip of the left lobe which is reduced in Fgfr1;2-DKO lungs. Epithelial branch number in the Fgfr1;2-DKO lung is comparable to that of normal at E11.5, but is reduced at E12.5. In all figures, solid horizontal lines at the bottom of representative panels indicate relative scales of samples shown. Each mutant lung is shown at the same magnification as corresponding normal control.
Figure 2. Inactivation of FGF9 signaling in lung mesenchyme leads to ectopic SMA-expressing cells
(A–D) In E12.5
Fgfr1;2-DKO lungs, the expression of Pea3 and Spry4 as detected by RNA in situ is reduced but not abolished. (E–P) Confocal images of antibody stained lungs. Colors are as labeled. Boxed areas in E, H, K, M are magnified in F/G, I/J, L and N, respectively. Insets in E and H are transverse section in the proximal lung showing that in the mutant, SMA-positive cells remain restricted to the parabronchial region, similar to the normal pattern. Arrowheads in J and P indicate ectopic SMA or SMM-positive cells near distal epithelial tips in the posterior mesenchyme, while arrowheads in H and I indicate ectopic SMA-positive cells near distal epithelial tips in other regions of the lung.
Figure 3. Ectopic SMCs arise independent of P-D patterning defects
(A–D) Confocal images of antibody stained lungs. Colors are as labeled. In the
Fgfr1;2-DKO lung, ectopic SMA-positive cells are present, and the epithelial branching pattern remains normal. (E, F) BrdU analysis in lung sections. (G) Diagram illustrating that at E11.75 there is no statistically significant difference in cell proliferation in Fgfr1;2-DKO lungs compared to control. (H–K) Immunofluorescent staining in lung sections. J and K are adjacent sections of H and I, respectively. SP-C (green) is expressed in the distal epithelium of Fgfr1;2-DKO lungs, similar to control lungs. Arrowheads indicate ectopic SMA- or SMM-positive cells adjacent to distal epithelium in Fgfr1;2-DKO lungs. (L–S) By RNA in situ hybridization, Sox2 and Sox9 are both expressed in a normal pattern in Fgfr1;2-DKO lungs as shown in wholemount (L-O,P,R) and in sections (Q,S). Arrowheads indicate the distal extent of the proximal Sox9 expression domain. Note that the proximal expression domain of Sox9 remains in the proximal mesenchyme, while the distal expression domain of Sox9 remains in the distal epithelium.
Figure 4. Inactivation of FGF9 signaling leads to ectopic airway SMCs
(A–D) By RNA in situ, in E12.5
Fgfr1;2-DKO lungs, Heyl is expressed in vascular SMCs, and is absent from the distal mesenchyme, similar to its pattern in control lungs. (E–H) Noggin expression as analyzed by β-gal staining in Noggin lungs. In E12.5 lacZ Fgf9 lungs, in addition to the normal pattern in parabronchial mesenchyme, β-gal activity is also detected in the distal mesenchyme (arrowheads in H). Boxed areas in A, C, E and G are magnified in B, D, F and H, respectively. −/−;Noggin lacZ/+
Figure 5. Downregulation of FGF signaling leads to reduced airway SMC progenitors
(A–D) Endothelial cells are labeled by anti-PECAM antibody. Boxed areas in A and C are magnified in B and D, respectively. Endothelial cells are reduced in
Fgfr1;2-DKO lungs compared to control. (E) Schematics of possible outcomes of ectopic SMC lineage experiment using Tek-Cre; Rosa-Gfp lungs. In outcome 1 following SU5402 treatment, there is overlap of endothelial lineage (labeled by GFP, green) and SMA expression (red, with the combination being yellow), suggesting that there is differentiation of endothelial cells into SMCs. In outcome 2 following SU5402 treatment, there is no overlap of green GFP-expressing cells and red SMA-expressing cells, indicating that ectopic SMCs do not arise from the endothelial lineage. (F–I) E11.5 Tek-Cre; Rosa-Gfp lungs after 24 hours of culture. Boxed areas in F and H are magnified in G and I, respectively. In SU5402-treated lungs, ectopic SMA-expressing cells in the distal mesenchyme (arrowhead in I) are not GFP-positive. (J–O) Fgf10 and Tbx4 expression as assayed by RNA in situ hybridization. Boxed area in J and L are magnified in K and M, respectively. As indicated by arrowheads, Fgf10 expression is reduced in the mutant. Tbx4 expression is also slightly reduced in the mutant. (P) Quantification of relative gene expression in E12.5 Fgfr1;2-DKO lungs compared to normal control by real-time RT-PCR analysis.
Figure 6. Inactivation of
Fgfr1;2 results in decreased WNT signaling activity and ectopic Myocardin expression
(A–F) Gene expression as detected by RNA in situ hybridization in E12.5 lungs. In
Fgfr1;2-DKO lungs, the expression of Wnt2 and Lef1 is reduced. Arrowhead in F indicates ectopic Myocardin expression in the posterior distal mesenchyme where ectopic SMA-positive cells are most frequently observed. Ventral views are shown in all panels.
Figure 7. FGF9 and SHH pathways act in parallel to control SMC differentiation
(A) Three possible mechanisms to illustrate how FGF9 and SHH signaling pathways may coordinate to regulate SMC differentiation. (B–E, H–M) Confocal compositions of lungs with SMCs labeled by anti-SMA antibody (red) and epithelium labeled by anti-ECAD antibody (green). (B–E) E11.75 lungs cultured in the presence or absence of cyclopamine (cycl). Arrowheads indicate the distal boundary of SMA expression. Cycl treatment leads to a reduced SMA-positive domain in both control and
Fgfr1;2-DKO lungs. (F,G) By RNA in situ hybridization, Ptch1 expression is reduced in the E12.5 Fgfr1;2-DKO lung compared to normal control. (H,I) E11.5 Shh mutant lungs cultured in the presence of SU5402 exhibit a larger SMA-positive domain than those cultured in the absence of SU5402. Open arrowheads indicate SMCs that are likely residual from esophageal tissue. Solid arrowheads indicate ectopic SMCs. (J–M) E12.5 lungs of indicated genotypes. Compared to normal control lung, SMCs are present in both the parabronchial and distal mesenchyme of −/− Fgf9 single mutant lung, absent in the Shh single mutant lung, and present in a disorganized pattern in the Fgf9;Shh double mutant lung.
Figure 8. A model of the role of FGF9 signaling in airway SMC development
Diagram of a distal lung unit with one epithelial branch encased in mesenchyme and mesothelium. FGF9 expressed by the mesothelium and distal epithelium signals to FGFRs expressed in the mesenchyme. The range of FGF9 signaling is indicated by the blue shaded region in the distal mesenchyme. Airway SMC progenitors constitute a subset of the cells in the distal mesenchyme. FGF9 signals to distal mesenchymal cells to suppress airway SMC differentiation and promote proliferation. As a consequence of cell proliferation, some mesenchymal cells are translocated out of the FGF9 signaling range. These cells can then differentiate into SMCs in response to inductive cues from the epithelium.
All figures (8)
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Carrier Proteins / genetics
Carrier Proteins / metabolism
Cell Differentiation / physiology*
Fibroblast Growth Factor 9 / genetics
Fibroblast Growth Factor 9 / metabolism*
Lung* / anatomy & histology
Myocytes, Smooth Muscle / cytology
Myocytes, Smooth Muscle / physiology*
Nuclear Proteins / genetics
Nuclear Proteins / metabolism
Receptor, Fibroblast Growth Factor, Type 1 / genetics
Receptor, Fibroblast Growth Factor, Type 1 / metabolism
Receptor, Fibroblast Growth Factor, Type 2 / genetics
Receptor, Fibroblast Growth Factor, Type 2 / metabolism
Signal Transduction / physiology*
Trans-Activators / genetics
Trans-Activators / metabolism
Fibroblast Growth Factor 9
Receptor, Fibroblast Growth Factor, Type 1
Receptor, Fibroblast Growth Factor, Type 2
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