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First published online 15 March 2006
doi: 10.1242/dev.02313

Development 133, 1507-1517 (2006)
Published by The Company of Biologists 2006
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FGF9 and SHH signaling coordinate lung growth and development through regulation of distinct mesenchymal domains

Andrew C. White1, Jingsong Xu2, Yongjun Yin1, Craig Smith1, Gregory Schmid1 and David M. Ornitz1,*

1 Department of Molecular Biology and Pharmacology, Washington University Medical School, St Louis, MO 63110, USA.
2 Brigham and Women's Hospital, Division of Critical Care and Pulmonary Medicine, Boston, MA 02115, USA.


Figure 1
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  		Fig. 1. FGF9 induces mesenchymal and epithelial expansion in vitro. (A-C) Increased distal mesenchyme adjacent to an FGF9 bead (red line, B) and decreased distal mesenchyme adjacent to an FGF10 bead (red line, C) compared with a BSA bead (red line, A) in E11.5 lung organ cultures incubated for 24 hours. (D-F) After 48 hours, epithelial luminal expansion occurs in cultures incubated with either FGF9 (E) or FGF10 (F) beads compared with control (D). Mesenchymal expansion at 48 hours (red lines, D-F) remained higher in explants treated with FGF9 (red lines, A-C). Arrow in E indicates a narrow band of mesenchyme between the epithelium and the FGF9 bead, not evident in FGF10 bead-treated cultures (F). (G) lacZ staining for the Rosa26 reporter demonstrates Dermo1-Cre activity throughout E13.5 lung mesenchyme. (H-K) Decreased lung size in mesenchymal Fgfr conditional knockouts at E17.5. Dermo1-Cre;Fgfr1+/–;Fgfr2+/– (H) and Dermo1-Cre;Fgfr1–/–;Fgfr2+/– (not shown) lungs exhibit wild-type size and morphology. Dermo1-Cre;Fgfr1+/–;Fgfr2–/– lungs (I) are moderately hypoplastic, and Dermo1-Cre;Fgfr1–/–;Fgfr2–/– lungs are severely hypoplastic with variable morphology (J,K). H-K are left lung lobes.

 

Figure 2
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  		Fig. 2. FGF9 induces mesenchymal and epithelial expansion in vivo. (A-D) Bi-transgenic TRE-Fgf9-IRES-eGfp;SPC-rtTA lungs from E11.5 and E12.5 embryos induced for 48 hours with doxycycline (Fgf9dox(48)) (B,D) exhibit increased overall size, an extended mesenchymal area and a reduction in branching compared with controls (A,C). (E,F) eGFP fluorescence in control (E) and Fgf9dox(48) (F) lung epithelium. (G,H) E14.5, Fgf9dox(48) lungs (H) demonstrated an increased size and expanded epithelial lumens. (I,J) Whole-mount in situ hybridization demonstrates Fgf9 epithelial expression in E14.5 Fgf9dox(48) lungs (J), consistent with eGFP fluorescence (F). Fgf9 expression and eGFP fluorescence was not seen in single TRE-Fgf9-IRES-eGfp lungs (E,I). G-J are left lung lobes.

 

Figure 3
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  		Fig. 3. Histology and mesenchymal differentiation in Fgf9dox(48) developing lungs. (A,B) Hematoxylin and Eosin stained sections showed increased size and mesenchymal area in E11.5 Fgf9dox(48) lungs (B). (C,D) Tie2-lacZ staining for endothelial cells in E12.5 Fgf9dox(48) lungs showing a multi-layered vascular network extending into the expanded mesenchyme (D), in contrast to a single layer of endothelial cells in wild-type controls (C). (E,F) Anti-PECAM immunohistochemistry confirmed an extended domain of blood vessels in E13.5 Fgf9dox(48) lungs (F). (G,H) Increased luminal space, fewer epithelial airways and a wide band of mesenchyme separating epithelial ducts was found in E14.5 Fgf9dox(48) lungs (H). (I,J) Smooth muscle actin (SMA) staining in proximal sub-epithelial mesenchyme adjacent to airways (a) is absent in E14.5 Fgf9dox(48) lungs (arrow, J). SMA was still found adjacent to blood vessels (bv). C, D and G-J are left lobes; E and F are cranial lobes.

 

Figure 4
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  		Fig. 4. FGF9 positively regulates HH signaling. (A,B) Whole-mount in situ hybridization demonstrates reduced Shh expression in E11.5 Fgf9–/– distal lung epithelium (box, B) compared with controls (A). Attached esophagus (e), by contrast, retained a similar level of expression in Fgf9–/– and wild-type controls. (C,D) Ptch1 expression was downregulated in E11.5 Fgf9–/– distal sub-epithelial mesenchyme (D). Esophageal expression of Ptch1, however, was not changed. (E-H) Increased Shh and sub-epithelial Ptch1 expression both distally and proximally in E11.5 Fgf9dox(48) lungs (F,H), compared with the distally restricted expression in controls (E,G). (I-P) FGF9 regulation of Shh and SHH signaling (Ptch1 expression) at E12.5 is similar to E11.5 (A-H). I-L are left lobes.

 

Figure 5
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  		Fig. 5. FGF9 induces sub-mesothelial mesenchymal proliferation independent of SHH signaling. (A-C) Two histologically distinct regions of mesenchymal cells are evident at E13.5. A population of sub-epithelial mesenchymal cells (SEM) wrap around the epithelial ducts, and a loose network of non-oriented sub-mesothelial mesenchymal cells (SMM) inhabit the area between the mesothelium and the SEM (A). In Fgf9–/– lungs, the SMM is largely absent (B), whereas a large expanse of SMM is present in Fgf9dox(48) lungs (C). (D-G) Whole-mount immunohistochemistry with anti-phosphohistone H3 (PH3) identified an increase in PH3-labeled cells in E11.5 lung explants incubated with FGF9 for 24 hours (E), compared with BSA-treated controls (D). Cyclopamine-only treated explants showed decreased overall proliferation (G). Explants incubated with both FGF9 and cyclopamine (F) appeared similar to FGF9-only treated explants (E) at low magnification. At higher magnification, cyclopamine-treated cultures (F',G') showed reduced cell proliferation in the SEM, whereas FGF9 increased proliferation in the SMM (E',F'). Explants treated with both cyclopamine and FGF9 (Cy/F9) have a combined effect, with increased PH3 labeling in the SMM and decreased labeling in the SEM (F9). (H-K) Staining for active caspase 3 demonstrates high levels of apoptosis in cyclopamine-treated explants (K), compared with relatively few labeled cells in BSA or FGF9-treated explants (H,I). A moderate amount of active caspase 3 staining is evident in the SEM of Cy/F9-treated explants, indicating a partial rescue of apoptosis by FGF9 (J). (L,M) Quantification of cell proliferation (PH3 labeled nuclei/unit area) in explants treated with FGF9 and cyclopamine (Cy) for 24 (L) and 48 (M) hours. Student t-test, ***P<0.0001; **P<0.001; *P<0.01.

 

Figure 6
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  		Fig. 6. Inhibition of HH signaling enhances epithelial budding. (A,B) Whole-mount in situ hybridization showing enhanced Ptch1 expression in the sub-epithelial mesenchyme following incubation with FGF9 (B), and inhibition of expression when incubated with cyclopamine (C). (D) Quantified decrease in distal epithelial buds in explants incubated with FGF9 and increased buds in explants treated with cyclopamine. (E-G) FGF9 treated E12.5 explants showing epithelial expansion and reduced branching (F) compared with controls. Cyclopamine-treated explants (G) exhibit increased epithelial budding (three versus two terminal branches; arrowheads in E,G). Lung epithelium is labeled with an antibody to TTF1. Student's t-test, n=2 or 3 explants for each condition; **P<0.003; *P<0.02.

 

Figure 7
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  		Fig. 7. FGF9 signaling regulates mesenchymal FGF gene expression and signaling. (A,B) Whole-mount in situ hybridization demonstrating upregulated and broad expression of Fgf10 in the sub-mesothelial mesenchyme of E13.5 Fgf9dox(48) lungs (B) compared with focal expression in control lungs (arrowhead, A). (C,D) Increased Fgf7 expression in both proximal and distal mesenchyme of Fgf9dox(48) lungs (D) compared with low level mesenchymal expression in control lungs (C). (E,F) Upregulation of Spry2 in epithelium of E11.5 whole lung explant cultures incubated for 48 hours with an FGF9 bead (F) compared with distally restricted expression in BSA controls (E). (G,H) Decreased Spry2 expression in E14.5 Fgf9–/– lungs (H) compared with controls (G). (I,J) Increased distal and ectopic proximal epithelial expression of Bmp4 in E14.5 Fgf9dox(48) lungs (J) compared with distally restricted expression in controls (I). (K,L) Comparable Bmp4 expression in E12.5 Fgf9–/– (L) and control lung epithelium (K), mirroring Fgf10 expression at this time (Colvin et al., 2001Go). (M,N) Decreased Bmp4 expression in E14.5 Fgf9–/– lungs (N) compared with controls (M). Bead location in E,F is indicated by orange circles. (A,B) Cranial lobes; (C,D) left lobes; (E,F) left lobes of explant cultures; (G-J,M,N) caudal lobes.

 

Figure 8
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  		Fig. 8. Model for mesothelial-mesenchymal-epithelial regulation of pseudoglandular stage lung development. Molecular pathways mediating Fgf9 loss- and gain-of-function phenotypes (A) and spatial relationships between molecules (B-E). In wild-type tissue (C), FGF9 maintains proliferation and Fgf10 expression in sub-mesothelial mesenchyme (1) (see Figs 5, 7) (Colvin et al., 2001Go), and at early stages induces Shh in the epithelium and SHH signaling in the adjacent sub-epithelial mesenchyme (2) (Fig. 4). SHH signaling is necessary for sub-epithelial mesenchyme survival and proliferation, and limits Fgf10 expression primarily in the interbud regions (3) (Fig. 5) (Pepicelli et al., 1998Go), allowing focal sources of Fgf10 to induce epithelial branching (4) (Bellusci et al., 1997bGo; Park et al., 1998Go; Weaver et al., 2000Go). In the absence of Fgf9, Fgf10 expression is reduced such that epithelial branching arrests at ~E12.5 (B) (Colvin et al., 2001Go). When Fgf9 is overexpressed in airway epithelium at later stages (E12.5-E14.5) (D), Fgf10 expression is increased in sub-mesothelial mesenchyme (Fig. 7), but probably repressed in sub-epithelial mesenchyme through enhanced HH signaling (Fig. 4), facilitating tubule elongation, but not branching (2, 3). Fgf7 expression is increased in Fgf9 overexpressing lungs throughout sub-epithelial mesenchyme, inducing rapid epithelial luminal dilation (5) (Fig. 7). Cyclopamine mediated repression of SHH signaling (E) is suggested to de-represses Fgf10 in the sub-epithelial mesenchyme leading to increased budding (Fig. 6).

 

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© The Company of Biologists Ltd 2006