Fetal and postnatal lung defects reveal a novel and required role for Fgf8 in lung development - PubMed (original) (raw)

Fetal and postnatal lung defects reveal a novel and required role for Fgf8 in lung development

Shibin Yu et al. Dev Biol. 2010.

Abstract

The fibroblast growth factor, FGF8, has been shown to be essential for vertebrate cardiovascular, craniofacial, brain and limb development. Here we report that Fgf8 function is required for normal progression through the late fetal stages of lung development that culminate in alveolar formation. Budding, lobation and branching morphogenesis are unaffected in early stage Fgf8 hypomorphic and conditional mutant lungs. Excess proliferation during fetal development disrupts distal airspace formation, mesenchymal and vascular remodeling, and Type I epithelial cell differentiation resulting in postnatal respiratory failure and death. Our findings reveal a previously unknown, critical role for Fgf8 function in fetal lung development and suggest that this factor may also contribute to postnatal alveologenesis. Given the high number of premature infants with alveolar dysgenesis and lung dysplasia, and the accumulating evidence that short-term benefits of available therapies may be outweighed by long-term detrimental effects on postnatal alveologenesis, the therapeutic implications of identifying a factor or pathway that can be targeted to stimulate normal alveolar development are profound.

Copyright © 2010 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Fgf8 hypomorphic mutants die at birth with dysplastic, hypercellular lungs

A–D) Whole mount E18.5 control (Fgf8 H/+) and hypomorph (Fgf8 H/−) littermate mice (A, B) and lungs (C, D) photographed side by side; note that in spite of the overall decrease in body size of the mutant, the lungs are as large as those in controls (vertical black lines in C and D are the same size). E–F′) Low (E, F) and high (E′, F′) magnification photographs of H&E stained paraffin sections from P0.5 inflation-fixed lung preparations. Note the hypercellular, thickened septal walls (compare red arrows in E′ and F′) and location of many red blood cells (pink stained cells, located in capillaries) embedded in the septa whereas in the control the red blood cells are immediately adjacent to the airspaces. TRU; terminal respiratory unit

Figure 2

Figure 2. Fgf8 is expressed at low levels throughout prenatal lung development and increases markedly postnatally

Quantitative reverse transcription-PCR was used to assay Fgf8 (A) and Fgf10 (B) transcript levels in lung tissue prepared from E11.5-P5 specimens. Fold expression is shown relative to that at E14.5 and normalized to gapdh. Fgf10 quantitation is shown for comparison and increases gradually over this time period. Ct; cycle threshold value

Figure 3

Figure 3. Separation of epithelial and mesenchymal cells from E15.5 lungs reveals enrichment of Fgf8 transcripts in the epithelial compartment

Immunocytochemistry using the antibodies/markers listed at top to assay isolated mesenchymal A–D) and epithelial (A′–D′) lung cells. Note that SMA and PECAM are detected as expected in mesenchymal cells (A and B, respectively) but not in epithelial cells (A′, B′) while Nkx2.1 and FoxA2 label epithelial cells. These staining patterns validate the stringency and specificity of the cell isolation method. E) Quantitative reverse transcription-PCR was used to compare transcript levels in the isolated mesenchymal and epithelial cells; Fgf8 transcripts were 6.5 fold greater in the epithelial cells. Other marker transcripts used as controls were present in the expected cellular compartments.

Figure 4

Figure 4. Excess proliferation occurs in lungs of Fgf8 hypomorphic mutants after E15.5

A) Immunohistochemical detection of BrdU-labeled nuclei (brown staining) in lung sections at the ages noted at top (E13.5–18.5). Control specimens are shown in the top row and Fgf8 hypomorphic specimens in the bottom row. There is a dramatic increase in the number of labeled cells in the mesenchyme and epithelia from E16.5 onward (red boxed panels). Note that the morphology of the mutant lung also appears normal prior to E16.5. B) Quantitation of distal mesenchymal proliferation confirms statistically significant increases in number of BrdU labeled nuclei in mutants at E16.5. ** p<0.005 C) Quantitation of epithelial cell proliferation confirms statistically significant increases in number of BrdU labeled nuclei in mutants at E16.5. ** p<0.005 D) Fraction of total (mesenchymal + epithelial) BrdU labeled nuclei is significantly increased in mutants at both E17.5 and E18.5. ** p<0.005

Figure 5

Figure 5. FGF8 stimulates BrdU incorporation by E15.5 lung mesenchymal cells in culture but represses it in epithelial cells

A, B) Bar graphs representing percentage of BrdU positive nuclei under the culture conditions listed. A) FGF8 stimulates BrdU incorporation by cultured E15.5 lung mesenchymal cells in the presence or absence of serum (a, b; c,d; e, f). ** p < 0.001 B) FGF8 represses BrdU incorporation by cultured E15.5 lung epithelial cells in the presence of serum (c, d and g, h). ** p < 0.001, * p < 0.01 C, D) Overall adherence, growth and survival of cultured E15.5 lung cells as reflected by the number of total cells present at the time of assay represented as a percent of the number initially plated. C) Fewer mesenchymal cells are present at 24 hours when cultured in the presence of serum regardless of the presence of FGF8 (compare a to c, and b to d) but these cells then proliferate over the next 24 hours to increase the total number of cells by 1.5 fold (g, h). D) Few epithelial cells adhere or survive to 24 hours in the absence of serum and FGF8 treatment slightly improves viability in the first 24 hours in the absence of serum (a versus, b). Serum dramatically improves the number of cells present at 24 hours although these numbers are still < 50% of that achieved by mesenchymal cells and FGF8 has no additive effect to serum (c, d). There are significantly fewer cells in the FGF8 treated serum-free group at 48 hours (e versus f) and this is remedied by treatment with serum (h) ** p < 0.001, * p < 0.01

Figure 6

Figure 6. Pathway-based heat maps of gene expression changes in E18.5 hypomorphic lung tissue relative to controls

Green symbolizes decreased expression in mutant and red, increased. The four columns in each heat map represent the results from each of the four different experiments and show that the expression changes detected were highly reproducible from array to array. The glucocorticoid map shows genes that have been previously shown to be dysregulated in glucocorticoid receptor null mutant lungs (Bird et al., 2007)). ECM, extracellular matrix; CAM, cell adhesion molecule

Figure 7

Figure 7. Loss of Fgf8 function in the epithelia of Fgf8;Isl1Cre conditional mutants phenocopies the lung phenotypes of Fgf8 hypomorphs but ablation in the lung mesenchyme of Fgf8;MesP1Cre mutants does not

A–D′) H&E stained paraffin sections from E18.5 lung specimens obtained from mutants with the genotypes listed at top. Low and high magnification views are shown and were obtained at similar peripheral locations in the lung. B, B′) Ablation of Fgf8 only in the mesenchyme with MesP1Cre permits normal lung development through fetal development. C, C′) Fgf8 hypomorphic mutant lung phenotype. D, D′) Fgf8;Isl1Cre conditional mutant lung has thickened septa and few airspaces, phenocopying the hypomorphic lung. E) The ROSA26 lacz Cre reporter shows that MesP1Cre is active and recombines in all lung mesenchymal (Me) precursors by E11.5 but no evidence of recombination is detectable in the lung epithelia (Ep). F) The ROSA26 lacz Cre reporter shows that Isl1Cre is active and recombines broadly in lung epithelial precursors by E11.5, but only in a subset of lung mesenchyme.

Figure 8

Figure 8. Excess proliferation occurs in the lungs of Fgf8;Isl1Cre conditional mutants after E15.5

A) Immunohistochemical detection of BrdU-labeled nuclei (brown DAB staining) in lung sections at the ages noted at top (E14.5–18.5). Control specimens are shown in the top row and Fgf8;Isl1Cre conditional mutant specimens in the bottom row. There is an excess in the number of labeled cells in the mesenchyme and epithelia from E16.5 onward (red boxed sections). Note morphology of the mutant lung also appears normal prior to E16.5. B) Quantitation of distal epithelial proliferation confirms statistically significant increases in number of BrdU labeled nuclei in mutants at E16.5. Note that proliferation of both cell types is increased in the controls in this background as compared to that of the controls in Figure 4. * p< 0.05 C) Quantitation of mesenchymal cell proliferation confirms statistically significant increases in number of BrdU labeled nuclei in mutants at E16.5. ** p<0.005 D) Fraction of total (mesenchymal + epithelial) BrdU labeled nuclei is significantly increased in mutants at both E17.5 and E18.5. ** p<0.005

Figure 9

Figure 9. Differentiation and vascular remodeling are disrupted at the saccular and early alveolar stages in Fgf8;Isl1Cre conditional mutants

All panels show immunohistochemical stains of cryosectioned lung specimens at the ages stated. A–C′) Staining for FoxA2 protein in the distal epithelia (red) from E16.5–e18.5. There are few FoxA2+ cells lining the forming airspaces in E17.5 and E18.5 controls (B, C white arrowheads) and a normal pattern in mutants at E16.5 (A′). At E17.5 (B′) and 18.5 (C′) mutants have a paucity of airspaces and persistent high numbers of FoxA2+ cells (yellow arrowheads) in the developing airspaces. D–F′) Staining of the developing pulmonary vasculature for PECAM immunoreactivity (green) reveals grossly normal vascular development in the mutants until E18.5; at E18.5 there is less detectable staining (F′, yellow arrowheads). G–H) Consistent with the FoxA2 data, double staining for Nkx2.1 (green) and nuclei (blue) shows a few Nkx2.1+ cells located in the septa, but not lining the airspaces of controls (white arrowheads) while there is abnormal persistence of many Nkx2.1+ cells lining the small airspaces of the mutants (red arrowheads). I, J) Double staining for surfactant protein C (SPC, red) and nuclei (blue) shows increased number of SPC+ Type II alveolar cells or a more primitive precursor cell lining the airspaces. K–N) Analysis at E19. Double staining for PECAM (yellow/green) and nuclei (red) reveals that, relative to controls (K, K′), there is a paucity of distal vessels and vessels are aberrantly located with the thickened septa of the mutants (L, L′) rather than adjacent to the airspaces. Aquaporin 5 staining reveals paucity of Type I cells lining the airspaces and their aberrant cuboidal morphology in mutants (N, arrowheads) as compared to their larger surface area and squamous morphology which gives a fine reticular pattern in controls (M, arrowheads).

Figure 10

Figure 10. Differentiation and vascular remodeling are disrupted at the saccular and early alveolar stages in Fgf8 hypomorphic mutants

All panels show immunohistochemical stains of cryosectioned lung specimens at the E18.5. A–A′) Staining of the developing pulmonary vasculature for PECAM immunoreactivity (red) reveals is less detectable staining in the mutants. B, B′) Double staining for surfactant protein C (SPC, red) and nuclei (blue) shows increased number of SPC+ cells lining the airspaces. C, C′) Double staining for Nkx2.1 (green) and nuclei (blue) shows rare Nkx2.1+ cells in the airspaces of controls (white arrowheads) while there is abnormal persistence of many Nkx2.1+ cells lining the small airspaces of the mutants. D, D′) Staining for FoxA2 protein in the distal epithelia (pink). There are few FoxA2+ cells lining the forming airspaces in controls while mutants have a paucity of airspaces and persistent high numbers of FoxA2+ cells lining the small airspaces. E, E′) Aquaporin 5 staining reveals paucity of Type I cells in mutants.

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