FGF9 and SHH Signaling Coordinate Lung Growth and Development

RESEARCH ARTICLE 1507 Development 133, 1507-1517 (2006) doi:10.1242/dev.02313 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,* Morphogenesis of the lung is regulated by reciprocal signaling between epithelium and mesenchyme. In previous studies, we have shown that FGF9 signals are essential for lung mesenchyme development. Using Fgf9 loss-of-function and inducible gain-of- function mouse models, we show that lung mesenchyme can be divided into two distinct regions: the sub-mesothelial and sub- epithelial compartments, which proliferate in response to unique growth factor signals. Fibroblast growth factor (FGF) 9 signals from the mesothelium (the future pleura) to sub-mesothelial mesenchyme through both FGF receptor (FGFR) 1 and FGFR2 to induce proliferation. FGF9 also signals from the epithelium to the sub-epithelial mesenchyme to maintain SHH signaling, which regulates cell proliferation, survival and the expression of mesenchymal to epithelial signals. We further show that FGF9 represses peribronchiolar smooth muscle differentiation and stimulates vascular development in vivo. We propose a model in which FGF9 and SHH signals cooperate to regulate mesenchymal proliferation in distinct submesothelial and subepithelial regions. These data provide a molecular mechanism by which mesothelial and epithelial FGF9 directs lung development by regulating mesenchymal growth, and the pattern and expression levels of mesenchymal growth factors that signal back to the epithelium. KEY WORDS: Fibroblast growth factor 9 (FGF9), Sonic hedgehog (SHH), Lung development, Branching morphogenesis, Mesothelium, Epithelium, Mesenchyme, Mouse INTRODUCTION normal lung development is not clear (Bellusci et al., 1997b; The embryonic lung develops from three distinct tissue layers: the Cardoso et al., 1997; Guo et al., 1996; Park et al., 1998; Post et al., outer mesothelium, the mesenchyme and the bronchial epithelium. 1996; Simonet et al., 1995; Tichelaar et al., 2000). Spry2, an Following formation of the primary epithelial branches that define inducible inhibitor of FGF and EGF receptor tyrosine kinase the lobular structure of the lung, morphogenesis proceeds to the signaling, is expressed in the distal epithelial tips and is functionally pseudoglandular stage (E9.5-E16), in which continued downstream of the mesenchymal FGF genes, acting to suppress mesenchymal growth is coupled with epithelial branching to create mesenchymal FGF signaling to epithelium (Hanafusa et al., 2002; the respiratory tree. During this crucial developmental stage, a Mailleux et al., 2001; Tefft et al., 2002; Tefft et al., 1999; Zhang et dynamic network of signaling growth factors interact across the al., 2001). three tissue boundaries to regulate lung growth and branching, FGF9 fulfills the role of a reciprocal epithelial to mesenchymal orchestrating proportional growth of the proximal conducting signal in the lung. At E10.5, Fgf9 is expressed in both the airways, the terminal epithelial buds and the surrounding vascular mesothelial lining (future pleura) of the lung bud and in the network (Shannon and Hyatt, 2004). epithelium of the developing airways. As lung development Several studies have identified fibroblast growth factors (FGFs) progresses through the pseudoglandular stage, Fgf9 expression that signal across epithelial and mesenchymal boundaries to regulate persists in the mesothelium but can no longer be detected in the lung both the pseudoglandular and subsequent stages of lung epithelium (Colvin et al., 1999). Fgf9–/– mice have severe lung development (Shannon and Hyatt, 2004). In vitro studies have hypoplasia and die in the perinatal period. The most characteristic shown that mesenchymally expressed FGF10 activates the ‘b’ splice feature of Fgf9–/– lungs is a reduced ratio of mesenchyme to form of FGF receptor 2 (FGFR2b) in lung epithelium to direct epithelium caused by a reduction in mesenchymal (but not budding by stimulating epithelial cell migration and proliferation epithelial) proliferation at E10.5-E11.5. In addition, a secondary (Bellusci et al., 1997b; Park et al., 1998; Peters et al., 1992; Weaver consequence of Fgf9 gene inactivation is reduced branching of the et al., 2000). Consistent with this activity, loss of function of Fgf10 epithelial tubules after ~E12.5. Previous studies suggested that a or Fgfr2b results in the absence of primary branching in vivo, molecular etiology of reduced branching morphogenesis may be causing the trachea to terminate as a blind sac (Arman et al., 1999; decreased mesenchymal Fgf10 at actively branching regions of the Celli et al., 1998; De Moerlooze et al., 2000; Min et al., 1998; Peters lung (Colvin et al., 2001). et al., 1995; Sekine et al., 1999). Fgf7, also a ligand for FGFR2b, is Sonic hedgehog (SHH) and bone morphogenic protein 4 (BMP4) T expressed in developing lung mesenchyme, but its function during also modulate lung mesenchymal and epithelial development. Bmp4 is expressed in the distal epithelium, where it appears to have a 1Department of Molecular Biology and Pharmacology, Washington University primary role in promoting distal epithelial differentiation and Medical School, St Louis, MO 63110, USA. 2Brigham and Women’s Hospital, Division antagonizing FGF-mediated epithelial budding, and has a suggested of Critical Care and Pulmonary Medicine, Boston, MA 02115, USA. role in specifying smooth muscle precursors (Bellusci et al., 1996; *Author for correspondence (e-mail: [email protected]) Bitgood and McMahon, 1995; Mailleux et al., 2005; Weaver et al., 2000). Additionally, Bmp4 is upregulated by FGF10 in vitro and in Accepted 30 January 2006 vivo (Lebeche et al., 1999; Mailleux et al., 2005; Mailleux et al., DEVELOPMEN 1508 RESEARCH ARTICLE Development 133 (8) 2001; Weaver et al., 2000). Shh is expressed in the distal epithelium Whole mount in situ hybridization, lacZ staining and of the lung throughout the pseudoglandular stage and binds to its immunohistochemistry receptor, patched 1 (Ptch1), in the adjacent sub-epithelial Whole-mount in situ hybridization was performed as described previously LacZ+/– mesenchyme (Bellusci et al., 1997a; Bitgood and McMahon, 1995; (Colvin et al., 2001). Tie2 and Noggin-lacZ expression was visualized by staining whole lobes with 1 mg/ml X-gal, 2 mM MgCl2, 35 mM Miller et al., 2001; Weaver et al., 2003). Other members of this ϫ signaling pathway, such as Gli1 and Hip1, colocalize with Ptch1 Ke3F(CN)6, 35 mM Ke4F(CN)6 and 1 PBS at room temperature in the dark. Following adequate color reaction, tissues were washed and (Chuang et al., 2003; Grindley et al., 1997). Both Shh gain-of- dehydrated to methanol. Tissues were then sectioned in paraffin wax at 5 function and loss-of-function mutations, as well as combinatorial ␮M (Tie2LacZ+/–) or cryosectioned at 12 ␮M (Noggin-lacZ) and Gli loss-of-function mutations, demonstrate that SHH signaling counterstained with Eosin. Panel comparisons were paired with littermate positively affects both mesenchymal and epithelial growth, as well controls, ensuring similar developmental time points, tissue processing and as lobe formation (reviewed by van Tuyl and Post, 2000). SHH is staining incubation periods (8-10 hours). proposed to modulate the epithelial branching pattern by focally For whole-mount immunohistochemistry, dissected lung tissues were suppressing Fgf10 in distal mesenchyme and upregulating Fgf7 fixed overnight in 4% PFA, dehydrated to 100% methanol and stored at (Lebeche et al., 1999; Pepicelli et al., 1998). Conversely, FGF10 –20°C. All incubations and washes were performed at 4°C while shaking. does not affect Shh, whereas high levels of FGF7 suppresses both Tissues were rehydrated, incubated with a blocking solution (2% skim milk, Shh expression and signaling (Lebeche et al., 1999; Park et al., 1% sheep serum, 0.1% Triton X-100) for 2 hours, primary and secondary antibodies, in blocking solution, overnight at 4°C and then washed (2% skim 1998). Although Shh appears unaffected at late stages of branching –/– milk, 0.1% Triton X-100) five times at hourly intervals. Visualization of in Fgf9 lungs (Colvin et al., 2001), we hypothesized that FGF9 secondary antibody conjugated to HRP was performed using the DAB kit may directly or indirectly regulate SHH signaling at early stages of (Vector Laboratories), according to manufacturer’s instructions. PECAM1- branching, which in turn may affect mesenchymal proliferation and stained tissues were sectioned in paraffin wax at 5 ␮M and counterstained the level and pattern of Fgf10. with Hematoxylin. Rat anti-PECAM1 (BD) was incubated at 1:500, To investigate further the mechanisms by which FGF9 regulates monoclonal anti-TTF1 (DakoCytomation) at 1:200, monoclonal anti- lung development, we have developed two gain-of-function models: phosphohistone H3 (Sigma) at 1:500 and rabbit anti-active caspase 3 (R&D) an in vivo method to transiently express ectopic FGF9 in lung at 1:600. Secondary HRP conjugated anti-mouse antibody (Chemicon) was incubated at 1:300, HRP anti-rat at 1:200 and HRP anti-rabbit at 1:400. epithelium, and in vitro organ culture that mimics FGF9 ␮ overexpression in the mesothelium. Combined with the Fgf9 loss- Immunohistochemistry on paraffin embedded 5 M sections was performed using a standard protocol with citrate buffer antigen retrieval. Rat anti-SMA of-function mouse, these two systems facilitated an examination

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