- Email: [email protected]
Fgf18 is required for embryonic lung alveolar development
BBRC Biochemical and Biophysical Research Communications 322 (2004) 887–892 www.elsevier.com/locate/ybbrc
Fgf18 is required for embryonic lung alveolar developmentq Hiroko Usuia, Masaki Shibayamaa, Norihiko Ohbayashia, Morichika Konishia, Shinji Takadab, Nobuyuki Itoha,* a
Department of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Sakyo, Kyoto 606-8501, Japan b Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi 444-8585, Japan Received 23 July 2004
Abstract Fgf18 is abundantly expressed in mouse embryonic lungs. To elucidate the roles of Fgf18 in mouse embryonic lung development, we examined the Fgf18/ embryonic lungs. Although the sizes of the Fgf18/ lungs were a little smaller in appearance than those of wild-type lungs, neither proximal nor distal airway branching in the Fgf18/ lungs was impaired. However, the Fgf18/ lungs at E18.5 had reduced alveolar space, thicker interstitial mesenchymal compartments, and many embedded capillaries. Cell proliferation in the Fgf18/ lungs was also transiently reduced around E17.5, although the expression of marker genes for lung epithelial cells in the Fgf18/ lungs was not impaired. The present findings indicate that the Fgf18 plays roles in lung alveolar development during late embryonic lung development stages. The cell proliferation during the terminal saccular stage stimulated by Fgf18 might play roles in the remodeling of the distal lung. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Fgf; Fgfr; Mouse; Lung; Alveolar space; Development; Morphogenesis; Proliferation; Differentiation
Lung development in mice is initiated as a ventral outpouching of endodermal cells from the anterior foregut into the surrounding mesenchyme at E9.5, and is further continued by branching morphogenesis and differentiation of specialized cells [1,2]. Lung development is dependent upon reciprocal interactions between epithelial cells and mesenchymal cells. These interactions are mediated by growth and differentiation factors including members of the Fgf family, Shh, and members of the Tgf-b family [1,2].
q Abbreviations: Fgf, fibroblast growth factor; Shh, sonic hedgehog; Tgf-b, transforming growth factor-b; Bmp, bone morphogenetic protein; E, embryonic day; BrdU, 5-bromo-2 0 -deoxyuridine; Fgfr, fibroblast growth factor receptor; CCSP, Clara cell secretory protein; SP-C, surfactant protein C; Aqp5, aquaporin 5; TTF-1, thyroid transcription factor-1; SP-A, surfactant protein A. * Corresponding author. Fax: +81 75 753 4600. E-mail address: [email protected] (N. Itoh).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.198
Fgfs are polypeptide growth factors with various biological activities both in vivo and in vitro, and play important roles in development and metabolism. The human/mouse Fgf family consists of 22 members [3]. Fgf signaling plays important roles in lung morphogenesis. Fgf10 plays crucial roles in lung branching morphogenesis during early embryonic lung development stages [4,5]. Fgf9 regulates lung epithelial airway branching and organ size, coordinating with Fgf10 [6]. Fgf18/ mice die shortly after birth. The phenotype of Fgf18/ mice indicates that Fgf18 plays crucial roles in both osteogenesis and chondrogenesis [7,8]. However, Fgf18 is also abundantly expressed in both embryonic and postnatal lungs [9]. In addition, in Fgf18 transgenic mice that over-expressed Fgf18 in the epithelial cells of the developing lungs, peripheral lung tubules were markedly diminished in number, whereas the size and extent of conducting airways were increased [10]. These results indicate that Fgf18 might play roles in lung development. However, the roles of Fgf18 in lung
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development remain to be elucidated. Therefore, we examined the phenotype of Fgf18/ embryonic lungs. Here, we report that Fgf18 plays roles in lung alveolar development but not lung branching morphogenesis. The roles of Fgf18 in embryonic lung development are quite distinct from those of Fgf10 and Fgf9. Materials and methods Fgf18/ mice. Generation of Fgf18/ mice and analysis of their genotypes were performed as described [7]. Analysis of airway branching. Tracheas of mouse embryos (E18.5) were injected with a 50:1 mixture of Mercox CL-2X resin and catalyst (Ladd Research Industries, Burlington, VT) and the plastic was allowed to solidify at room temperature for 15 min. The lungs were incubated at 50 °C for 15 min in water, incubated at room temperature for 3 h in 20% KOH, and washed with water several times. In situ hybridization. The mouse embryonic lungs were frozen in powdered dry ice, and their sections were cut at 16 lm with a cryostat, thaw-mounted onto poly-L -lysine-coated slides, and stored at 85 °C until hybridization. 35S-labeled mouse antisense RNA probes were transcribed using T7 RNA polymerase or SP6 RNA polymerase with uridine 5 0 -a-[35S]thiotriphosphate (30 TBq/mmol) (Amersham Biosciences, Buckinghamshire, UK). The sections were examined by in situ hybridization with the labeled probe, followed by dipping in liquid emulsion (NTB3) (Eastman Kodak, Rochester, NY) diluted 1:1, and exposed for 3–4 weeks as described. The sections were counterstained with hematoxylin and eosin. Silver grains were visualized by dark-field microscopy. Histological analysis by light microscopy. The mouse embryos were fixed overnight in BouinÕs solution, dehydrated, and embedded in paraffin. Sections (5 lm) were cut from fixed embryos and stained with hematoxylin and eosin. Histological analysis by electron microscopy. The left lobes of embryonic lungs were minced into 0.5-mm cubes and were prefixed in 2% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C. After washing the cubes in 0.1 M cacodylate buffer at 4 °C, the cubes were postfixed in 2% osmium tetroxide and were dehydrated in graded ethanol. After dehydration, the cubes were substituted with propylene oxide and then with propylene oxide and epoxy resin. After substitution, the cubes were embedded into the gelatin capsule with epoxy resin at 60 °C for 2 days. Ultrathin sections (70–80 nm) were prepared by an ultramicrotome (LKB-8800) (LKB-Produckter, Bromma, Sweden) and stained with 2% uranyl acetate and then with lead. The sections were observed with a JEM-200EX transmission electron microscope (JEOL, Tokyo, Japan). Analysis of BrdU incorporation. Pregnant mice were intraperitoneally injected with BrdU (Amersham Biosciences) (100 mg/kg body weight) 1.5 h before being killed. The embryonic lungs were fixed overnight in BouinÕs fixative at 4 °C, dehydrated through an ethanol series, cleared in xylene, embedded in paraffin, and sectioned at 5 lm. Sections were incubated with anti-BrdU antibody using a cell proliferation kit (Amersham Biosciences). Anti-BrdU antibody was detected using a Vectastain ABC system with biotinylated anti-mouse IgG antibody (Vector, Burlingame, CA). After detection of BrdU-positive nuclei, sections were counterstained with nuclear fast red.
Fig. 1. Morphological comparison of wild-type and Fgf18/ mouse embryonic lungs. Lungs were dissected from wild-type (A) and Fgf18/ (B) mice at E18.5. The individual lobes of the wild-type lung (C–G) and the Fgf18/ lung (H–L). Right cranial lobe (C,H). Right median lobe (D,I). Right caudual lobe (E,J). Left lobe (F,K). Accessory lobe (G,L). WT, wild-type; /, Fgf18/.
lungs had the correct number of lobes, although the sizes of their lobes were a little smaller in appearance than those of the wild-type lungs (Fig. 1). We examined the lungs from at least five independent litters. The sizes of all the Fgf18/ lungs were a little smaller than those of the wild-type and Fgf18+/ lungs. The preservation of number and orientation of lobes in the Fgf18/ lungs suggest that airway branching was not impaired in those lungs. Morphological and in situ hybridization analysis of Fgf18/ lung branching morphogenesis We examined airway branching in the Fgf18/ lungs at E18.5 using plastic epithelial casts. Neither proximal nor distal airway branching was impaired in the Fgf18/ lungs (data not shown). These results are essentially consistent with those described above (Fig. 1). Fgf10, Bmp4, and Shh were shown to play crucial roles in lung branching morphogenesis [11,12]. We also examined the expression of Fgf10, Bmp4, and Shh in embryonic lungs at E15.5 by in situ hybridization. As expected, no apparent difference in the expression was observed between the wild-type and Fgf18/ lungs (data not shown).
Results Morphological analysis of Fgf18/ lungs
Histological analysis of Fgf18/ lungs by light and electron microscopy
The overall gross phenotype of the embryonic lungs (E18.5) of Fgf18/ mice was examined. The Fgf18/
We examined embryonic lungs at E12.5, E16.5, and E18.5 by analysis using a light microscope with hema-
H. Usui et al. / Biochemical and Biophysical Research Communications 322 (2004) 887–892
Fig. 2. Histological analysis of mouse embryonic lungs by light microscopy. Mouse embryonic lungs at E12.5 (A,B), E16.5 (C,D), and E18.5 (E–H) were examined by histological analysis with hematoxylin and eosin staining. Wt, wild-type lungs (A,C,E,G); /, Fgf18/ lungs (B,D,F,H). Closed and open arrowheads (G,H) indicate capillaries closely abutting alveolar spaces and embedded capillaries, respectively.
toxylin and eosin staining. Essentially, no apparent phenotypic difference could be observed between the wildtype and Fgf18/ lungs at E12.5 and E16.5 (Figs. 2A–D). However, a clear difference was observed in the embryonic lungs at E18.5. Fgf18/ lungs had reduced alveolar spaces and thicker interstitial mesenchymal compartments (Figs. 2E–H). Although most capillaries closely abutted alveolar spaces in the wildtype lungs, many capillaries in the Fgf18/ lungs were embedded. Embryonic lungs at E18.5 were also examined by electron microscopic analysis (Fig. 3). In the wild-type lungs, most capillaries closely abutted alveolar spaces. In contrast, significant morphological abnormality was observed in the Fgf18/ lungs. The alveolar spaces were greatly shrunk. Many capillaries were embedded. These results are essentially consistent with those by light microscopic analysis. Expression of lung epithelial markers in Fgf18/ lungs As the histological analysis indicated that the distal epithelial differentiation in Fgf18/ lungs might be impaired, we examined the expression of lung epithelial markers in embryonic lungs at E18.5 (Fig. 4). CCSP is a marker for Clara epithelial cells of the proximal epithelium [13]. SP-C and Aqp5 are markers for alveolar type 2 and type 1 cells of the distal epithelium, respectively [13]. However, there was essentially no obvious difference in their expression between the wild-type
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Fig. 3. Histological analysis of embryonic lungs by electron microscopy. Mouse embryonic lungs at E18.5 were examined by transmission electron microscopy. Asterisks indicate capillaries. WT, wild-type; /, Fgf18/. Scale bar, 5 lm.
and Fgf18/ lungs. In addition, we also examined the expression of TTF-1, a lung mesenchymal marker, SP-A, a lung epithelial cell marker, and T1-a, an alveolar type 1 cell marker. There was essentially no obvious difference in their expression as well (data not shown). Cell proliferation in Fgf18/ lungs We also examined proliferation of embryonic lung cells by BrdU immunohistochemistry at E14.5–E18.0. No obvious difference in the proportion of BrdU-positive cells (40–50%) was observed in either mesenchymal or epithelial cells between the wild-type and Fgf18/ lungs at E14.5 (data not shown). After E16.5, the proportion in wild-type lungs was gradually decreased (Fig. 5B). These results were essentially consistent with those reported [1,2]. No obvious difference in the proportion in mesenchymal cells between the wild-type and Fgf18/ lungs at E16.5 and E17.0 was observed. However, the proportion in epithelial cells was significantly or greatly decreased in the Fgf18/ lungs at E16.5 or E17.0, respectively (Figs. 5A and B). As it was difficult to distinguish between epithelial and mesenchymal cells at E17.5 and E18.0, all the BrdU-positive cells were compared to the total number of cells. The proportion of BrdU-positive cells in the Fgf18/ lungs at E17.5 was greatly decreased compared with that
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Fig. 4. Expression of lung epithelial marker genes in mouse embryonic lungs. The expression of lung epithelial marker genes in embryonic lungs at E18.5 was examined by in situ hybridization. The sections were counterstained with hematoxylin and eosin. Bright-field (A,C,E,G,I,K) and dark-field (B,D,F,H,J,L) photographs are shown. White grains in the dark-field photograph show their expression. CCSP, a marker for Clara epithelial cells of the proximal epithelium (A–D). SP-C, a marker for alveolar type 2 cells of the distal epithelium (E–H). Aqp5, a marker for alveolar type 1 cells of the distal epithelium (I–L). WT, wild-type (A,B,E,F,I,J); /, Fgf18/ (C,D,G,H,K,L). Scale bar, 500 lm.
of the wild-type lungs (Figs. 5A and B). A weak but significant difference in the lungs at E18.5 was also observed.
Expression of Fgf18 and Fgfr2c in mouse embryonic lungs We examined the expression of Fgf18 in mouse embryonic lungs at E14.5, E16.5, and E17.5 (Fig. 6). At E14.5, Fgf18 was expressed in the mesenchyme surrounding the airway epithelium. However, Fgf18 was widely expressed in the lungs at E16.5 and E17.5. Fgf18 can bind to and activate Fgfr2C and Fgfr3c among the Fgfrs [14,15]. A null mutation of Fgfr2 results in embryonic lethality at E4 [16]. In contrast, Fgfr3/ mice are viable. They suffer skeletal dysplasias but no lung dysplasia [17]. These results indicate that a potential receptor for Fgf18 in embryonic lungs is Fgfr2c. Therefore, we also examined the expression of Fgfr2C in the embryonic lungs. At E14.5, Fgfr2c was expressed in the airway epithelium. However, Fgfr2C was widely expressed in the lungs at E16.5 and E17.5 (Fig. 6).
Discussion
Fig. 5. Proliferation of lung cells. (A) Proliferation of embryonic lung cells was examined by BrdU immunohistochemistry. Lungs at E17.0 (a,b) and E17.5 (c,d). WT, wild-type lungs (a,c); /, Fgf18/ lungs (b,d). Scale bar, 20 lm. (B) Proliferation of embryonic lung cells at E16.5, E17.0, E17.5, and E18.5 was examined by BrdU immunohistochemistry. The proportion of BrdU-positive cells in the cells of the wild-type (solid bars) and Fgf18/ lungs (open bars) is shown. Results are means ± SD for three independent sections.
Lung development in mice is initiated as a ventral outpouching of endodermal cells from the anterior foregut into the surrounding mesenchyme at E9.5, and is further continued by branching morphogenesis and differentiation of specialized cells through three phases [1,2]. During the pseudoglandular phase (E9.5–E16), the lungs undergo repeated dichotomous branching to form respiratory bronchioles and alveolar ducts. During the canalicular phase (E16–E17), the rapid growth rate diminishes, dichotomous branching is completed, and differentiation of the epithelial cells lining the ducts begins to take place. During the terminal saccular phase (E17 to term), there is continued growth of capillaries and remodeling of the distal lung to resemble adult lung
H. Usui et al. / Biochemical and Biophysical Research Communications 322 (2004) 887–892
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Fig. 6. Expression of Fgf18 and Fgfr2c in mouse embryonic lungs. The expression of Fgf18 and Fgfr2c in mouse embryonic lungs at E14.5, E17.5, and E18.5 was examined by in situ hybridization. The sections were counterstained with hematoxylin and eosin. Bright-field (A,C,E,G,I,K) and darkfield (B,D,F,H,J,L) photographs are shown. White grains in the dark-field photograph show their expression. Scale bar, 200 lm.
parenchyma. This remodeling involves the continued development of the capillary network, cellular differentiation, thinning of mesenchyme-derived stroma, and expansion of presumptive alveoli [1,2]. Lung development is dependent upon reciprocal interactions between epithelial cells and mesenchymal cells. These interactions are mediated by growth and differentiation factors including members of the Fgf family, Shh, and members of the Tgf-b family [1,2]. Fgf10/ mice completely lack lungs, indicating that mesenchymal Fgf10 is essential for epithelial branching in the developing lung [4,5]. Fgf9/ mice exhibit lung hypoplasia [6]. The Fgf9/ lungs exhibit reduced mesenchyme and decreased branching of airways. Fgf10 signaling from the mesenchyme and reciprocal Fgf9 signaling from the epithelium coordinately regulate epithelial airway branching and organ size in lung development [6]. Both Fgf10 and Fgf9 play crucial roles in lung development during the pseudoglandular phase. The peripheral lung tubules of Fgf18 transgenic mice that over-expressed Fgf18 in the epithelial cells of the developing lungs were markedly diminished in number, whereas the size and extent of the conducting airways were increased. These results indicate that Fgf18 is capable of enhancing proximal and inhibiting peripheral airway formation [10]. However, the Fgf18/ lungs had the correct number of lobes, and the examination of airway branching in the Fgf18/ lungs using plastic epithelial casts showed that neither proximal nor distal airway branching was impaired in the Fgf18/ lungs. Furthermore, the expression of marker genes for branching morphogenesis, including Fgf10, Bmp4, and Shh, was not impaired in the Fgf18/ lungs. These results indicate that Fgf18 plays essentially no role in lung branching morphogenesis. Histological analysis of the Fgf18/ lungs by both light and electron microscopy revealed that the Fgf18/ lungs at E18.5 had reduced alveolar spaces, thicker interstitial mesenchymal compartments, and
many embedded capillaries. However, there was essentially no obvious difference in the expression of lung epithelial markers, including CCSP, SP-C, and Aqp5, between the wild-type and Fgf18/ lungs. These results indicate that Fgf18 plays roles in the remodeling of the distal lung during the terminal saccular stage. As described above, the sizes of the Fgf18/ lungs were only a little smaller in appearance than those of the wild-type lungs. During the pseudoglandular phase (E9.5–E16), the lungs rapidly grow by undergoing repeated dichotomous branching to form respiratory bronchioles and alveolar ducts [1,2]. As expected, no difference in the proliferation activity in either mesenchymal or epithelial cells was observed between the wild-type and Fgf18/ lungs during the pseudoglandular phase. However, the proliferation activity in cells of the Fgf18/ lungs was transiently decreased during the terminal saccular phase. The proliferation stimulated by Fgf18 might play roles in the remodeling of the distal lung during the terminal saccular stage. Fgf18 signaling in the development is potentially mediated by activation of Fgfr2c, although Fgf10 signaling and Fgf9 signaling in the development are mediated by activating Fgfr2b and Fgfr1c, respectively [4–6]. In conclusion, in addition to Fgf10 and Fgf9, Fgf18 is also an Fgf crucial to embryonic lung development. Fgf18 plays roles in the proliferation of lung cells and the remodeling of the distal lung, mainly during the later (terminal saccular) phase. The roles of Fgf18 in embryonic lung development are quite distinct from those of Fgf10 and Fgf9 that play crucial roles in embryonic lung development, mainly during the earlier (pseudoglandular) phase.
Acknowledgments This work was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Cul-
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ture, Sports, Science and Technology, Japan, and by a grant from the Takeda Science Foundation, Japan.
References [1] C. Mendelson, Role of transcription factors in fetal lung development and surfactant protein gene expression, Annu. Rev. Physiol. 62 (2000) 875–915. [2] D. Warburton, S. Bellusci, P.M. Del Moral, V. Kaartinen, M. Lee, D. Tefft, W. Shi, Growth factor signaling in lung morphogenetic center: automaticity, stereotypy and symmetry, Respir. Res. 4 (2003) 5. [3] D.M. Ornitz, N. Itoh, Fibroblast growth factors, Genome Biol. 2 (2001) 3005.1–3005.12. [4] H. Min, D.M. Danilenko, S.A. Scully, B. Bolon, B.D. Ring, J.E. Tarpley, M. DeRose, W.S. Simonet, Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless, Genes Dev. 12 (1998) 3156– 3161. [5] K. Sekine, H. Ohuchi, M. Fujiwara, M. Yamasaki, T. Yoshizawa, T. Sato, N. Yagishita, D. Matsui, Y. Koga, N. Itoh, S. Kato, Fgf10 is essential for limb and lung formation, Nat. Genet. 21 (1999) 138–141. [6] J.S. Colvin, A.C. White, S.J. Pratt, D.M. Ornitz, Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme, Development 128 (2001) 2095–2106. [7] N. Ohbayashi, M. Shibayama, Y. Kurotaki, M. Imanishi, T. Fijimori, N. Itoh, S. Takada, FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis, Genes Dev. 16 (2002) 870–879.
[8] Z. Liu, J. Xu, J.S. Colvin, D.M. Ornitz, Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18, Genes Dev. 16 (2002) 859–869. [9] N. Ohbayashi, M. Hoshikawa, S. Kimura, M. Yamasaki, S. Fukui, N. Itoh, Structure and expression of the mRNA encoding a novel fibroblast growth factor, FGF-18, J. Biol. Chem. 273 (1998) 18161–18164. [10] J.A. Whitsett, J.C. Clark, L. Picard, J.W. Tischelaar, S.E. Wert, N. Itoh, A.K. Perl, M.T. Stahlman, Fibroblast growth factor 18 influences proximal programming during lung morphogenesis, J. Biol. Chem. 277 (2002) 22743–22749. [11] W.V. Cardoso, Molecular regulation of lung development, Annu. Rev. Physiol. 63 (2001) 471–494. [12] D.M. Warburton, Schwarz, D. Tefft, G. Flores-Delgado, K.D. Anderson, W.V. Cardoso, The molecular basis of lung morphogenesis, Mech. Dev. 92 (2000) 55–81. [13] W. Shu, Y.Q. Jiang, M.M. Lu, E.E. Morrisey, Wnt7b regulates mesenchymal proliferation and vascular development in the lung, Development 129 (2002) 4831–4842. [14] M. Hoshikawa, A. Yonamine, M. Konishi, N. Itoh, FGF-18 is a neuron-derived glial cell growth factor expressed in the rat brain during early postnatal development, Brain Res. Mol. Brain Res. 105 (2002) 60–66. [15] J. Xu, Z. Liu, D.M. Ornitz, Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures, Development 127 (2000) 1833–1843. [16] E. Arman, R. Haffner-Krausz, Y. Chen, J.K. Heath, P. Lonai, Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian develoment, Proc. Natl. Acad. Sci. USA 95 (1998) 5082–5087. [17] J.S. Colvin, B.A. Bohne, G.W. Harding, D.G. McEwen, D.M. Ornitz, Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3, Nat. Genet. 12 (1996) 390–397.
Fgf18 is required for embryonic lung alveolar developmentq Hiroko Usuia, Masaki Shibayamaa, Norihiko Ohbayashia, Morichika Konishia, Shinji Takadab, Nobuyuki Itoha,* a
Department of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Sakyo, Kyoto 606-8501, Japan b Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi 444-8585, Japan Received 23 July 2004
Abstract Fgf18 is abundantly expressed in mouse embryonic lungs. To elucidate the roles of Fgf18 in mouse embryonic lung development, we examined the Fgf18/ embryonic lungs. Although the sizes of the Fgf18/ lungs were a little smaller in appearance than those of wild-type lungs, neither proximal nor distal airway branching in the Fgf18/ lungs was impaired. However, the Fgf18/ lungs at E18.5 had reduced alveolar space, thicker interstitial mesenchymal compartments, and many embedded capillaries. Cell proliferation in the Fgf18/ lungs was also transiently reduced around E17.5, although the expression of marker genes for lung epithelial cells in the Fgf18/ lungs was not impaired. The present findings indicate that the Fgf18 plays roles in lung alveolar development during late embryonic lung development stages. The cell proliferation during the terminal saccular stage stimulated by Fgf18 might play roles in the remodeling of the distal lung. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Fgf; Fgfr; Mouse; Lung; Alveolar space; Development; Morphogenesis; Proliferation; Differentiation
Lung development in mice is initiated as a ventral outpouching of endodermal cells from the anterior foregut into the surrounding mesenchyme at E9.5, and is further continued by branching morphogenesis and differentiation of specialized cells [1,2]. Lung development is dependent upon reciprocal interactions between epithelial cells and mesenchymal cells. These interactions are mediated by growth and differentiation factors including members of the Fgf family, Shh, and members of the Tgf-b family [1,2].
q Abbreviations: Fgf, fibroblast growth factor; Shh, sonic hedgehog; Tgf-b, transforming growth factor-b; Bmp, bone morphogenetic protein; E, embryonic day; BrdU, 5-bromo-2 0 -deoxyuridine; Fgfr, fibroblast growth factor receptor; CCSP, Clara cell secretory protein; SP-C, surfactant protein C; Aqp5, aquaporin 5; TTF-1, thyroid transcription factor-1; SP-A, surfactant protein A. * Corresponding author. Fax: +81 75 753 4600. E-mail address: [email protected] (N. Itoh).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.198
Fgfs are polypeptide growth factors with various biological activities both in vivo and in vitro, and play important roles in development and metabolism. The human/mouse Fgf family consists of 22 members [3]. Fgf signaling plays important roles in lung morphogenesis. Fgf10 plays crucial roles in lung branching morphogenesis during early embryonic lung development stages [4,5]. Fgf9 regulates lung epithelial airway branching and organ size, coordinating with Fgf10 [6]. Fgf18/ mice die shortly after birth. The phenotype of Fgf18/ mice indicates that Fgf18 plays crucial roles in both osteogenesis and chondrogenesis [7,8]. However, Fgf18 is also abundantly expressed in both embryonic and postnatal lungs [9]. In addition, in Fgf18 transgenic mice that over-expressed Fgf18 in the epithelial cells of the developing lungs, peripheral lung tubules were markedly diminished in number, whereas the size and extent of conducting airways were increased [10]. These results indicate that Fgf18 might play roles in lung development. However, the roles of Fgf18 in lung
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H. Usui et al. / Biochemical and Biophysical Research Communications 322 (2004) 887–892
development remain to be elucidated. Therefore, we examined the phenotype of Fgf18/ embryonic lungs. Here, we report that Fgf18 plays roles in lung alveolar development but not lung branching morphogenesis. The roles of Fgf18 in embryonic lung development are quite distinct from those of Fgf10 and Fgf9. Materials and methods Fgf18/ mice. Generation of Fgf18/ mice and analysis of their genotypes were performed as described [7]. Analysis of airway branching. Tracheas of mouse embryos (E18.5) were injected with a 50:1 mixture of Mercox CL-2X resin and catalyst (Ladd Research Industries, Burlington, VT) and the plastic was allowed to solidify at room temperature for 15 min. The lungs were incubated at 50 °C for 15 min in water, incubated at room temperature for 3 h in 20% KOH, and washed with water several times. In situ hybridization. The mouse embryonic lungs were frozen in powdered dry ice, and their sections were cut at 16 lm with a cryostat, thaw-mounted onto poly-L -lysine-coated slides, and stored at 85 °C until hybridization. 35S-labeled mouse antisense RNA probes were transcribed using T7 RNA polymerase or SP6 RNA polymerase with uridine 5 0 -a-[35S]thiotriphosphate (30 TBq/mmol) (Amersham Biosciences, Buckinghamshire, UK). The sections were examined by in situ hybridization with the labeled probe, followed by dipping in liquid emulsion (NTB3) (Eastman Kodak, Rochester, NY) diluted 1:1, and exposed for 3–4 weeks as described. The sections were counterstained with hematoxylin and eosin. Silver grains were visualized by dark-field microscopy. Histological analysis by light microscopy. The mouse embryos were fixed overnight in BouinÕs solution, dehydrated, and embedded in paraffin. Sections (5 lm) were cut from fixed embryos and stained with hematoxylin and eosin. Histological analysis by electron microscopy. The left lobes of embryonic lungs were minced into 0.5-mm cubes and were prefixed in 2% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C. After washing the cubes in 0.1 M cacodylate buffer at 4 °C, the cubes were postfixed in 2% osmium tetroxide and were dehydrated in graded ethanol. After dehydration, the cubes were substituted with propylene oxide and then with propylene oxide and epoxy resin. After substitution, the cubes were embedded into the gelatin capsule with epoxy resin at 60 °C for 2 days. Ultrathin sections (70–80 nm) were prepared by an ultramicrotome (LKB-8800) (LKB-Produckter, Bromma, Sweden) and stained with 2% uranyl acetate and then with lead. The sections were observed with a JEM-200EX transmission electron microscope (JEOL, Tokyo, Japan). Analysis of BrdU incorporation. Pregnant mice were intraperitoneally injected with BrdU (Amersham Biosciences) (100 mg/kg body weight) 1.5 h before being killed. The embryonic lungs were fixed overnight in BouinÕs fixative at 4 °C, dehydrated through an ethanol series, cleared in xylene, embedded in paraffin, and sectioned at 5 lm. Sections were incubated with anti-BrdU antibody using a cell proliferation kit (Amersham Biosciences). Anti-BrdU antibody was detected using a Vectastain ABC system with biotinylated anti-mouse IgG antibody (Vector, Burlingame, CA). After detection of BrdU-positive nuclei, sections were counterstained with nuclear fast red.
Fig. 1. Morphological comparison of wild-type and Fgf18/ mouse embryonic lungs. Lungs were dissected from wild-type (A) and Fgf18/ (B) mice at E18.5. The individual lobes of the wild-type lung (C–G) and the Fgf18/ lung (H–L). Right cranial lobe (C,H). Right median lobe (D,I). Right caudual lobe (E,J). Left lobe (F,K). Accessory lobe (G,L). WT, wild-type; /, Fgf18/.
lungs had the correct number of lobes, although the sizes of their lobes were a little smaller in appearance than those of the wild-type lungs (Fig. 1). We examined the lungs from at least five independent litters. The sizes of all the Fgf18/ lungs were a little smaller than those of the wild-type and Fgf18+/ lungs. The preservation of number and orientation of lobes in the Fgf18/ lungs suggest that airway branching was not impaired in those lungs. Morphological and in situ hybridization analysis of Fgf18/ lung branching morphogenesis We examined airway branching in the Fgf18/ lungs at E18.5 using plastic epithelial casts. Neither proximal nor distal airway branching was impaired in the Fgf18/ lungs (data not shown). These results are essentially consistent with those described above (Fig. 1). Fgf10, Bmp4, and Shh were shown to play crucial roles in lung branching morphogenesis [11,12]. We also examined the expression of Fgf10, Bmp4, and Shh in embryonic lungs at E15.5 by in situ hybridization. As expected, no apparent difference in the expression was observed between the wild-type and Fgf18/ lungs (data not shown).
Results Morphological analysis of Fgf18/ lungs
Histological analysis of Fgf18/ lungs by light and electron microscopy
The overall gross phenotype of the embryonic lungs (E18.5) of Fgf18/ mice was examined. The Fgf18/
We examined embryonic lungs at E12.5, E16.5, and E18.5 by analysis using a light microscope with hema-
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Fig. 2. Histological analysis of mouse embryonic lungs by light microscopy. Mouse embryonic lungs at E12.5 (A,B), E16.5 (C,D), and E18.5 (E–H) were examined by histological analysis with hematoxylin and eosin staining. Wt, wild-type lungs (A,C,E,G); /, Fgf18/ lungs (B,D,F,H). Closed and open arrowheads (G,H) indicate capillaries closely abutting alveolar spaces and embedded capillaries, respectively.
toxylin and eosin staining. Essentially, no apparent phenotypic difference could be observed between the wildtype and Fgf18/ lungs at E12.5 and E16.5 (Figs. 2A–D). However, a clear difference was observed in the embryonic lungs at E18.5. Fgf18/ lungs had reduced alveolar spaces and thicker interstitial mesenchymal compartments (Figs. 2E–H). Although most capillaries closely abutted alveolar spaces in the wildtype lungs, many capillaries in the Fgf18/ lungs were embedded. Embryonic lungs at E18.5 were also examined by electron microscopic analysis (Fig. 3). In the wild-type lungs, most capillaries closely abutted alveolar spaces. In contrast, significant morphological abnormality was observed in the Fgf18/ lungs. The alveolar spaces were greatly shrunk. Many capillaries were embedded. These results are essentially consistent with those by light microscopic analysis. Expression of lung epithelial markers in Fgf18/ lungs As the histological analysis indicated that the distal epithelial differentiation in Fgf18/ lungs might be impaired, we examined the expression of lung epithelial markers in embryonic lungs at E18.5 (Fig. 4). CCSP is a marker for Clara epithelial cells of the proximal epithelium [13]. SP-C and Aqp5 are markers for alveolar type 2 and type 1 cells of the distal epithelium, respectively [13]. However, there was essentially no obvious difference in their expression between the wild-type
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Fig. 3. Histological analysis of embryonic lungs by electron microscopy. Mouse embryonic lungs at E18.5 were examined by transmission electron microscopy. Asterisks indicate capillaries. WT, wild-type; /, Fgf18/. Scale bar, 5 lm.
and Fgf18/ lungs. In addition, we also examined the expression of TTF-1, a lung mesenchymal marker, SP-A, a lung epithelial cell marker, and T1-a, an alveolar type 1 cell marker. There was essentially no obvious difference in their expression as well (data not shown). Cell proliferation in Fgf18/ lungs We also examined proliferation of embryonic lung cells by BrdU immunohistochemistry at E14.5–E18.0. No obvious difference in the proportion of BrdU-positive cells (40–50%) was observed in either mesenchymal or epithelial cells between the wild-type and Fgf18/ lungs at E14.5 (data not shown). After E16.5, the proportion in wild-type lungs was gradually decreased (Fig. 5B). These results were essentially consistent with those reported [1,2]. No obvious difference in the proportion in mesenchymal cells between the wild-type and Fgf18/ lungs at E16.5 and E17.0 was observed. However, the proportion in epithelial cells was significantly or greatly decreased in the Fgf18/ lungs at E16.5 or E17.0, respectively (Figs. 5A and B). As it was difficult to distinguish between epithelial and mesenchymal cells at E17.5 and E18.0, all the BrdU-positive cells were compared to the total number of cells. The proportion of BrdU-positive cells in the Fgf18/ lungs at E17.5 was greatly decreased compared with that
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Fig. 4. Expression of lung epithelial marker genes in mouse embryonic lungs. The expression of lung epithelial marker genes in embryonic lungs at E18.5 was examined by in situ hybridization. The sections were counterstained with hematoxylin and eosin. Bright-field (A,C,E,G,I,K) and dark-field (B,D,F,H,J,L) photographs are shown. White grains in the dark-field photograph show their expression. CCSP, a marker for Clara epithelial cells of the proximal epithelium (A–D). SP-C, a marker for alveolar type 2 cells of the distal epithelium (E–H). Aqp5, a marker for alveolar type 1 cells of the distal epithelium (I–L). WT, wild-type (A,B,E,F,I,J); /, Fgf18/ (C,D,G,H,K,L). Scale bar, 500 lm.
of the wild-type lungs (Figs. 5A and B). A weak but significant difference in the lungs at E18.5 was also observed.
Expression of Fgf18 and Fgfr2c in mouse embryonic lungs We examined the expression of Fgf18 in mouse embryonic lungs at E14.5, E16.5, and E17.5 (Fig. 6). At E14.5, Fgf18 was expressed in the mesenchyme surrounding the airway epithelium. However, Fgf18 was widely expressed in the lungs at E16.5 and E17.5. Fgf18 can bind to and activate Fgfr2C and Fgfr3c among the Fgfrs [14,15]. A null mutation of Fgfr2 results in embryonic lethality at E4 [16]. In contrast, Fgfr3/ mice are viable. They suffer skeletal dysplasias but no lung dysplasia [17]. These results indicate that a potential receptor for Fgf18 in embryonic lungs is Fgfr2c. Therefore, we also examined the expression of Fgfr2C in the embryonic lungs. At E14.5, Fgfr2c was expressed in the airway epithelium. However, Fgfr2C was widely expressed in the lungs at E16.5 and E17.5 (Fig. 6).
Discussion
Fig. 5. Proliferation of lung cells. (A) Proliferation of embryonic lung cells was examined by BrdU immunohistochemistry. Lungs at E17.0 (a,b) and E17.5 (c,d). WT, wild-type lungs (a,c); /, Fgf18/ lungs (b,d). Scale bar, 20 lm. (B) Proliferation of embryonic lung cells at E16.5, E17.0, E17.5, and E18.5 was examined by BrdU immunohistochemistry. The proportion of BrdU-positive cells in the cells of the wild-type (solid bars) and Fgf18/ lungs (open bars) is shown. Results are means ± SD for three independent sections.
Lung development in mice is initiated as a ventral outpouching of endodermal cells from the anterior foregut into the surrounding mesenchyme at E9.5, and is further continued by branching morphogenesis and differentiation of specialized cells through three phases [1,2]. During the pseudoglandular phase (E9.5–E16), the lungs undergo repeated dichotomous branching to form respiratory bronchioles and alveolar ducts. During the canalicular phase (E16–E17), the rapid growth rate diminishes, dichotomous branching is completed, and differentiation of the epithelial cells lining the ducts begins to take place. During the terminal saccular phase (E17 to term), there is continued growth of capillaries and remodeling of the distal lung to resemble adult lung
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Fig. 6. Expression of Fgf18 and Fgfr2c in mouse embryonic lungs. The expression of Fgf18 and Fgfr2c in mouse embryonic lungs at E14.5, E17.5, and E18.5 was examined by in situ hybridization. The sections were counterstained with hematoxylin and eosin. Bright-field (A,C,E,G,I,K) and darkfield (B,D,F,H,J,L) photographs are shown. White grains in the dark-field photograph show their expression. Scale bar, 200 lm.
parenchyma. This remodeling involves the continued development of the capillary network, cellular differentiation, thinning of mesenchyme-derived stroma, and expansion of presumptive alveoli [1,2]. Lung development is dependent upon reciprocal interactions between epithelial cells and mesenchymal cells. These interactions are mediated by growth and differentiation factors including members of the Fgf family, Shh, and members of the Tgf-b family [1,2]. Fgf10/ mice completely lack lungs, indicating that mesenchymal Fgf10 is essential for epithelial branching in the developing lung [4,5]. Fgf9/ mice exhibit lung hypoplasia [6]. The Fgf9/ lungs exhibit reduced mesenchyme and decreased branching of airways. Fgf10 signaling from the mesenchyme and reciprocal Fgf9 signaling from the epithelium coordinately regulate epithelial airway branching and organ size in lung development [6]. Both Fgf10 and Fgf9 play crucial roles in lung development during the pseudoglandular phase. The peripheral lung tubules of Fgf18 transgenic mice that over-expressed Fgf18 in the epithelial cells of the developing lungs were markedly diminished in number, whereas the size and extent of the conducting airways were increased. These results indicate that Fgf18 is capable of enhancing proximal and inhibiting peripheral airway formation [10]. However, the Fgf18/ lungs had the correct number of lobes, and the examination of airway branching in the Fgf18/ lungs using plastic epithelial casts showed that neither proximal nor distal airway branching was impaired in the Fgf18/ lungs. Furthermore, the expression of marker genes for branching morphogenesis, including Fgf10, Bmp4, and Shh, was not impaired in the Fgf18/ lungs. These results indicate that Fgf18 plays essentially no role in lung branching morphogenesis. Histological analysis of the Fgf18/ lungs by both light and electron microscopy revealed that the Fgf18/ lungs at E18.5 had reduced alveolar spaces, thicker interstitial mesenchymal compartments, and
many embedded capillaries. However, there was essentially no obvious difference in the expression of lung epithelial markers, including CCSP, SP-C, and Aqp5, between the wild-type and Fgf18/ lungs. These results indicate that Fgf18 plays roles in the remodeling of the distal lung during the terminal saccular stage. As described above, the sizes of the Fgf18/ lungs were only a little smaller in appearance than those of the wild-type lungs. During the pseudoglandular phase (E9.5–E16), the lungs rapidly grow by undergoing repeated dichotomous branching to form respiratory bronchioles and alveolar ducts [1,2]. As expected, no difference in the proliferation activity in either mesenchymal or epithelial cells was observed between the wild-type and Fgf18/ lungs during the pseudoglandular phase. However, the proliferation activity in cells of the Fgf18/ lungs was transiently decreased during the terminal saccular phase. The proliferation stimulated by Fgf18 might play roles in the remodeling of the distal lung during the terminal saccular stage. Fgf18 signaling in the development is potentially mediated by activation of Fgfr2c, although Fgf10 signaling and Fgf9 signaling in the development are mediated by activating Fgfr2b and Fgfr1c, respectively [4–6]. In conclusion, in addition to Fgf10 and Fgf9, Fgf18 is also an Fgf crucial to embryonic lung development. Fgf18 plays roles in the proliferation of lung cells and the remodeling of the distal lung, mainly during the later (terminal saccular) phase. The roles of Fgf18 in embryonic lung development are quite distinct from those of Fgf10 and Fgf9 that play crucial roles in embryonic lung development, mainly during the earlier (pseudoglandular) phase.
Acknowledgments This work was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Cul-
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ture, Sports, Science and Technology, Japan, and by a grant from the Takeda Science Foundation, Japan.
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