Fgf10 Dosage Is Critical for the Amplification of Epithelial Cell Progenitors and for the Formation of Multiple Mesenchymal Lineages During Lung Development

Developmental Biology 307 (2007) 237–247 www.elsevier.com/locate/ydbio

Fgf10 dosage is critical for the amplification of epithelial cell progenitors and for the formation of multiple mesenchymal lineages during lung development

Suresh K. Ramasamy a,1, Arnaud A. Mailleux b,1, Varsha V. Gupte a, Francisca Mata a, Frédéric G. Sala a, Jacqueline M. Veltmaat c, Pierre M. Del Moral a, Stijn De Langhe a, Sara Parsa a, Lisa K. Kelly d, Robert Kelly e, Wei Shia a, Eli Keshet f, Parviz Minoo g, ⁎ David Warburton a, Savério Bellusci a,

a Developmental Biology Program, Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA b Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA c Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore d Division of Pediatrics, Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA e Developmental Biology Institute of Marseille Luminy-UMR6216-CNRS-Université de la Méditerranée, France f Department of Molecular Biology, The Hebrew University–Hadassah Medical School, Jerusalem, Israel g Department of Pediatrics, Women's and Children's Hospital, USC Keck School of Medicine, Los Angeles, CA 90033, USA Received for publication 23 October 2006; revised 24 April 2007; accepted 26 April 2007 Available online 3 May 2007

Abstract

The key role played by Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit lung agenesis. However, Fgf10 is continuously expressed throughout lung development suggesting extended as well as additional roles for FGF10 at later stages of lung organogenesis. We previously reported that the enhancer trap Mlcv1v-nLacZ-24 transgenic mouse strain functions as a reporter for Fgf10 expression and displays decreased endogenous Fgf10 expression. In this paper, we have generated an allelic series to determine the impact of Fgf10 dosage on lung development. We report that 80% of the newborn Fgf10 hypomorphic mice die within 24 h of birth due to respiratory failure. These mutant mouse lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in TTF1 and SP-B expressing cells correlating with reduced epithelial cell proliferation and associated with a decrease in activation of the canonical Wnt signaling in the epithelium. Analysis of vascular development shows a reduction in PECAM expression at E14.5, which is associated with a simplification of the vascular tree at E18.5. We also show a decrease in α-SMA expression in the respiratory airway suggesting defective smooth muscle cell formation. At the molecular level, these defects are associated with decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelial/ mesenchymal ratio in the Fgf10 hypomorphic lungs. Thus, our results indicate that FGF10 plays a pivotal role in maintaining epithelial progenitor cell proliferation as well as coordinating alveolar smooth muscle cell formation and vascular development. © 2007 Elsevier Inc. All rights reserved.

Keywords: Fgf10 hypomorph; Mesenchymal differentiation; Smooth muscle cells; Lung emphysema; Vascularization

Introduction sites where prospective epithelial buds will appear. Moreover, its dynamic pattern of expression and its ability to induce epithelial Fibroblast growth factor 10 (FGF10) is responsible for directed expansion and budding in organ cultures have led to the outgrowth of the lung endoderm (Bellusci et al., 1997). In the hypothesis that FGF10 governs the directional outgrowth of developing lung, Fgf10 is expressed in the distal mesenchyme at lung buds during branching morphogenesis (Bellusci et al., 1997). Furthermore, FGF10 was shown to induce chemotaxis of the ⁎ Corresponding author. distal lung epithelium (Park et al., 1998; Weaver et al., 2000). E-mail address: [email protected] (S. Bellusci). Consistent with these observations, Fgf10 null mutants show 1 Contributed equally to this work. multiple organ defects including lung agenesis (Min et al., 1998;

Sekine et al., 1999), therefore hampering the study of subsequent desired stages and fixed in 4% PFA. Following fixation, the embryos were FGF10 role during lung development. FGF10 is the main ligand washed in 1× PBS and stained with X-gal solution. Stained lungs were sectioned (25μm) using a vibratome. for fibroblast growth factor receptor 2b (FGFR2b) during embryonic development as demonstrated by the remarkable Mutant embryos similarity of phenotypes exhibited by embryos where these genes have been inactivated (Min et al., 1998; Sekine et al., 1999; Burns The Mlc1v-nLacZ-24 line (called for simplification Mlc1v-LacZ or Mlc1v in et al., 2004; De Moerlooze et al., 2000; Mailleux et al., 2002; this paper) has been previously described (Kelly et al., 2001). The transgene containing an nLacZ reporter gene (containing a nuclear localization signal) is Burns et al., 2004; Del Moral et al., 2006a,b). Moreover, integrated upstream of the Fgf10 gene. Fgf10+/−;Mlc1v-LacZ+/− embryos were inhibition of FGFR2b signaling from embryonic day 14.5 generated by crossing Fgf10+/− and Mlc1v-LacZ+/− mice (Kelly et al., 2001; (E14.5) onwards using a transgenic mouse line expressing a Sekine et al., 1999) on a C57BL/6 background. Fgf10+/− littermates were used as − soluble FGFR2b (FGFR2b-HFc) under the control of an control embryos at different developmental stages. The Fgf10 and Mlc1v-LacZ+ inducible lung-specific, Surfactant protein C promoter (SpC- alleles were genotyped as described previously (Kelly et al., 2001; Mailleux et al., 2002). The number of Fgf10+/−;Mlc1v-LacZ+/− embryos used in this study (47 in rtTA), resulted in decreased epithelial morphogenesis before total) at the different stages was as follows: E12.5 (n=7), E13.5 (n=3), E14.5 birth and caused severe emphysema at maturity (Hokuto et al., (n=6), E16.5 (n=2), E17.5 (n=10); E18.5 (n=7); PN (n=12). 2003). Interestingly, Fgf10 expression level keeps increasing during lung development between E11.5 and E18.5 (Bellusci et Real-time RT–PCR al., 1997), suggesting that Fgf10 most likely plays an extended +/− +/− +/− and vital role during late lung organogenesis. Total RNA was extracted from individual Fgf10 and Fgf10 ;Mlc1v E14.5 embryonic lungs (n=3 for each genotype) using the RNeasy kit (Gibco We have previously shown that a transgenic mouse line with BRL) according to the manufacturer's instructions. DNA contaminations were the β-galactosidase gene under the control of Fgf10 regulatory removed in the total RNA using Turbo DNAse (Ambion). Total RNA was sequences can be used to monitor Fgf10 expression in the heart, reverse-transcribed using the Superscript-III first strand super mix (Invitrogen) lung and somites (Kelly et al., 2001; Mailleux et al., 2005; following the manufacturer's recommendations. 5 μg of the total RNA was used Veltmaat et al., 2006). Originally, Kelly et al. (2001) sought to to prepare cDNA from the isolated total RNA using oligodT primers. 25 pg cDNA was used for each of the real-time PCR reactions using the primers and express LacZ under the control of the myocardial ventricular- probes designed by the online Roche software: Probe finder version 2.20, https:// slow skeletal muscle Myosin Alkali-light chain (Mlc1v)promoter. www.roche-applied%1Escience.com/sis/rtpcr/upl/adc.jsp. All real-time PCR reac- In one of four founders, the expression pattern in the developing tions were performed with Roche: FastStart TaqMan® Probe Master kits, according heart suggested that LacZ was under the control of Fgf10 to the manufacturer's instructions in Roche Light Cycler 1.5 Real-Time PCR regulatory sequences. In addition, analysis of the integration site machine. 18S ribosomal RNA was used as an internal control for all analysis. showed that the Mlc1v-LacZ cassette had integrated 120 kb Analysis of SP-B and TTF-1 expression upstream of the Fgf10 gene. Based on the expression pattern as well as on the site of insertion, the authors proposed that LacZ Newborn pup lungs (3 Fgf10+/−;Mlc1v-LacZ+/− and 3 control) were fixed expression was under the control of Fgf10 regulatory sequences. overnight in paraformaldehyde, rinsed in PBS twice for 5 min, transferred to 70% We reported that the insertion of the Mlc1v-LacZ cassette ethanol overnight and stored in 100% ethanol. The samples were then embedded in paraffin and sections (5 μm) were cut. The number of cells expressing SP-B disrupted the endogenous expression of Fgf10 (Mailleux et al., and TTF1 was quantified using immunohistochemistry protocol with Envision+ +/− 2005). The Mlc1v-LacZ mice were crossed with Fgf10 mice to HRP system (Dakocytomation). Alternatively, expression of SP-B was also generate an allelic series to determine the effect of decreasing determined using in situ hybridization on sections using a specific mouse SP-B Fgf10 expression on parabronchial smooth muscle cell formation. probe (gift from Dr. Jeffrey Whitsett). Positive cells were scored in random We also demonstrated that Fgf10 identifies a new population of portions of a section in eight photomicrographs (50× magnification). A total number of 3000 cells were counted per sample. The results are presented as a ratio parabronchial smooth muscle cell progenitors located in the sub- of total number of positive cells/total number of cells. Lungs from three mutant mesothelial mesenchyme (Mailleux et al., 2005). and three control mice were taken at birth. In this paper, we report our analysis of Fgf10 hypomorphic (Fgf10+/−;Mlc1v-LacZ+/−) lungs at various developmental Antibodies stages. Newborn Fgf10 hypomorphic mice exhibit respiratory μ failure and consequently die within 48 h. Analysis shows The following antibodies were used for immunohistochemistry on 5 m thick paraffin sections: mouse monoclonal antibody against α-SMA (Sigma) at a severe lung hypoplasia, dilation of the distal airways and large dilution of 1/5000, mouse monoclonal antibody against Phospho-p44/42 MAPK hemorrhagic areas. Therefore, this mouse represents a unique (cell Signaling: 20G11) at a dilution of 1/50, mouse monoclonal antibody opportunity to study the role of FGF10 in coordinating against Thyroid Transcription factor-1 (NeoMarkers), rabbit polyclonal anti- epithelial morphogenesis with the differentiation of the body against Surfactant protein B (Chemicon), anti-PECAM/CD31 antibody mesenchyme, in particular with the formation of alveolar (BD Pharmage), anti-Laminin antibodies (Sigma), anti-Elastin polyclonal antibodies (EPC) at a dilution of 1/200. Slides were mounted with Vectashield smooth muscle cells and the vasculature. (Vector Labs) containing DAPI. Immunohistochemistry for β-catenin was performed with the Envision kit from Dako cytomation, β-catenin antibodies Materials and methods (BD Biosciences) was used at 1:100. Photomicrographs were taken using a Leica DMRA fluorescence microscope with a Hamamatsu Digital CCD Camera. Analysis of LacZ expression Visualization of the vascular system by plastic casting β-galactosidase activity was monitored in whole-mount and histological samples as described by Kelly et al. (1995). The day of vaginal plug was To visualize the vessels of the vascular system of E18.5 embryonic lung, we considered as embryonic day 0.5 (E0.5). Embryos were dissected in 1× PBS at used Batson's #17 Anatomical Corrosion Kit (Polysciences). The pregnant S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247 239 female mouse was euthanized according to our institute regulations. The 100% EtOH. The samples were then embedded in paraffin and sections (5 μm) abdominal area was opened and 5 U of heparin (1000 U/ml) was injected into the were cut. The PCNA staining kit (Zymed Labs Inc. 93-1143) was used for inferior vena cava. Embryos were then isolated, the sternum exposed and immunostaining. At E14.5, the total numbers of cells in the epithelium as well approximately 4 ml of a methyl methacrylate-based resin was injected into the as the number of PCNA positive cells in the epithelium were scored in three right ventricle of the heart. The embryos were cured for 3 h in ice-cold water and photomicrographs (64× magnification), in random portions of a section of were subsequently soaked in distilled water overnight at 4 °C. Embryos were three different mutant and control lungs each. A similar count was done for then digested in maceration solution using approximately three times volume of mesenchymal cells. At E17.5, as it was no longer possible to distinguish tissue being digested and were incubated at 50 °C for 72 h or until all tissue was between epithelium and mesenchyme, the total number of cells versus the total cleared. Maceration solution was changed every 3 h. Images of the cast were number of PCNA positive cells were counted for each genotype. A total documented using a Leica MZ125 with SPOT v3.2.0 digital camera. number of 3000 cells were counted per sample. The significance in proliferation between control and hypomorphic lungs was evaluated by one- Proliferation tailed paired t-test. P values less than 0.05 were considered to be statistically significant. Dehydrated E14.5 and E17.5 lungs (3 Fgf10+/−;Mlc1v-LacZ+/− and 3 controls for each stage) were fixed overnight in paraformaldehyde, rinsed In situ gelatinase assay twice in PBS for 5 min, transferred to 70% EtOH overnight and stored in In situ zymogram assays were carried out using fluorescein isothyocyanate labeled gelatin (Molecular Probes, Eugene, OR) to detect gelatinase activity (corresponding to MMP2 and MMP9 expressed at high levels in the smooth muscle cells). The reaction product was visualized using fluorescence microscopy.

Results

Insertion of the Mlc1v-LacZ cassette results in decreased endogenous Fgf10 expression

We recently published that the insertion of the Mlvc1v- LacZ cassette 120 kb upstream of the transcriptional start site of the Fgf10 gene, resulted in a general decrease in Fgf10 expression in the embryo (Mailleux et al., 2005; Veltmaat et al., 2006). We took advantage of this characteristic of the Mlc1v-LacZ line to generate an allelic series to determine the impact of Fgf10 dosage on lung development. Mlc1v-LacZ+/− line was crossed with Fgf10neo null allele heterozygote mice (denoted as Fgf10−)to generate Fgf10+/+;Mlc1v-LacZ−/− (wild type), Fgf10+/+; Mlc1v-LacZ+/−, Fgf10+/−;Mlc1v-LacZ−/− (called thereafter Fgf10+/−) and Fgf10+/−;Mlc1v-LacZ+/− (called also thereafter Fgf10 hypomorph) embryos at different developmental stages. As the lungs of embryos corresponding to the first three genotypes

Fig. 1. Fgf10+/−;Mlc1v-LacZ+/− lungs display reduction in Fgf10 expression and hypoplasia. (A) Nearly identical Cp values of ribosomal 18S messenger RNA (mRNA) indicate comparable amounts of starting material (Fgf10+/− Cp=20.26, Fgf10+/−;Mlc1v-LacZ+/− Cp=20.23). (B) Real-time PCR quantification in E14.5 lungs show a decrease of Fgf10 mRNA in Fgf10+/−;Mlc1v-LacZ+/− lungs (Fgf10+/− Cp=27.04, Fgf10+/−;Mlc1v-LacZ+/− Cp=27.53). Reduced epithelial branching in Fgf10+/−;Mlc1v-LacZ+/− vs. control (Fgf10+/−) lungs. (C–F) Fgf10+/− (control) lung of E12.0 with 4 buds in the left lobe shown at high magnification. (D) Fgf10+/−;Mlc1v-LacZ+/− lung at E12.0 with 3 buds in the left lobe. (E) Fgf10+/− (control) lung of E12.5 with 6 buds in the left lobe. (F) Fgf10+/−;Mlc1v-LacZ+/− lung at E12.5 with 4 buds in the left lobe. (G) Fgf10+/− (control) lung at E17.5. (H) Hypoplastic E17.5 Fgf10+/−;Mlc1v-LacZ+/− lung with absent the accessory lobe (white arrow). (I) Whole embryo body weights at E14.5, E17.5 and newborn (NB) of Fgf10+/− and Fgf10+/−;Mlc1v-LacZ+/− embryos show significant differences (475.83 mg, 994.40 mg, 1344 mg vs. 440 mg, 881.62 mg, 1188.85 mg, respectively). (J) Embryo/lung weight ratio at E14.5, E17.5 and newborn (NB) in control (Fgf10+/−) and Fgf10+/−; Mlc1v-LacZ+/− embryos show significant differences (0.029, 0.035, 0.034 vs. 0.017, 0.019, 0.017, respectively). 240 S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247

(Fgf10+/+;Mlc1v-LacZ−/− (wild type), Fgf10+/+;Mlc1v-LacZ+/− characterized by a decrease in epithelial branching (Figs. 1C– and Fgf10+/−;Mlc1v-LacZ−/− (or Fgf10+/−)) were phenotypically F). Consistent with our hypothesis that this phenotype results identical throughout all the developmental stages examined (data from reduced Fgf10 levels, a similar phenotype was observed not shown), we considered as control for this study, among these 3 in transgenic lungs over-expressing Sprouty2, a negative genotypes, the one corresponding to the lowest expression of regulator of FGF10 signaling (Mailleux et al., 2001). Primary Fgf10 (Fgf10+/−). 47 Fgf10+/+;Mlc1v-LacZ+/− compound he- lobe formation in the mutant lungs was generally not affected, terozygous embryos were generated at expected Mendelian ratios except for the accessory lobe, which was absent in 9 of the 52 and examined at different stages. As expected, real-time PCR Fgf10+/+;Mlc1v-LacZ+/− lungs generated (see arrow in Figs. experiments showed that Fgf10 expression is reduced by 27% in 1G, H). In addition, the remaining lobes in mutant lungs were E14.5 Fgf10+/−;Mlc1v-LacZ+/− lungs compared to corresponding substantially smaller in volume than their littermate controls. Fgf10+/− lungs (Figs. 1A,B; n=3, P=0.005). In order to rule out general growth retardation as the cause for the hypoplastic lungs, the body weight (Fig. 1I) and lung weight Fgf10+/−;Mlc1v-LacZ+/− compound heterozygous embryos (data not show) of control (Fgf10+/−) and Fgf10 hypomorphic exhibit lung hypoplasia embryos at different developmental stages were measured (the number of embryos examined were n=3 at E14.5, n=5 at E17.5 The Fgf10+/−;Mlc1v-LacZ+/− embryos displayed defects in and n=7 at birth, for control and mutant). While the body weight lung development. At E12 and E12.5, the lung phenotype was between control and mutant embryos were not significantly

Fig. 2. Fgf10+/−;Mlc1v-LacZ+/− lungs display decreased epithelial proliferation and reduction of canonical Wnt activation. (A–D) PCNA staining was used to monitor cell proliferation in Fgf10+/− and Fgf10+/−;Mlc1v-LacZ+/− at E14.5 and E17.5. Black arrow heads indicate PCNA labeled cells. (C) Note the specific decrease in proliferation of epithelium in mutant lungs at E14.5. (D) At E17.5, a general decrease in cell proliferation is observed in mutant lungs. (E–J) The TOPGAL allele was introduced in Fgf10+/+;Mlc1v-LacZ+/− and Fgf10+/−;Mlc1v-LacZ+/− lungs to determine the level of activation of canonical Wnt signaling at E13.5. In this experiment, β-gal expression indicates both Fgf10 expression in the mesenchyme (black arrowhead) and TOPGAL expression in the epithelium (white arrow). (E–F) LacZ staining is detected throughout the epithelium of E13.5 control lung (white arrow) indicating active Wnt signaling. (G) Vibratome section of the lung shown in panel E. (H) Decrease LacZ staining in Fgf10 hypomorphic lung epithelium (white arrow) indicates reduced Wnt signaling. Note the previously described increase in Fgf10/ LacZ expression in the mesenchyme (black arrowhead) corresponds to a defect in the engagement of the PSMC progenitors into the SMC lineage (Mailleux et al., 2005). (J) Vibratome section of the lung shown in panel H. (K, L) IHC for β-catenin in E12.5 control lung showing strong nuclear β-catenin expression in the epithelium. (M, N) IHC for β-catenin in E12.5 Fgf10 hypomorphic lung showing many blue nuclei in the epithelium indicating decreased nuclear β-catenin activation. e – epithelium, m – mesenchyme, wt – wild type (Fgf10+/−), mt – mutant. S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247 241 different at the given developmental stages (P=0.28 at E14.5; Pb0.05). Interestingly, mesenchymal proliferation is not P=0.05 at E17.5 and P=0.26 at birth), there was a clear affected in mutant (Fig. 2C; 5.6±1.4% vs. 5.4±1.2%, reduction in the lung weight of the mutant embryos compared to respectively). The decreased proliferation in the epithelium the littermate controls at the three stages examined (7.5 mg±2.8, is associated with a lower number of P-ERK positive cells in 16.8 mg± 2.1; 20,2 mg±0.6 vs. 13.7 mg±2.1, 34.8 mg±10.6, the Fgf10 hypomorphic lung epithelium (47% and 38% 45.7 mg±15 at E14.5, E17.5 and birth, respectively, Pb0.01 for decrease in the distal epithelium of Fgf10 hypomorph vs. all three stages examined). This observation is confirmed by the control lung at E13.5 and E14.5, respectively, n=3, data not calculation of normalized wet weight ratios (wet weight of the shown, Pb0.05). At E17.5, we could no longer distinguish lung divided by the wet weight of the embryo; 1.7±0.1%, 1.9± between epithelial and mesenchymal cells. A general reduc- 0.4% and 1.7±0.4% for the Fgf10+/−;Mlc1v-LacZ+/− mutants at tion in cell proliferation in the mutant is observed (32.8±8% E14.5, E17.5 and birth, respectively; versus 2.9±0.7%, 3.5± vs. 21.6±4.5%; n=3 for each genotype; Pb0.05). Wnt 0.7% and 3.4±1.0% for the corresponding control embryos or signaling is crucial during lung development to control the pups, respectively, Fig. 1J). The differences observed at each maintenance and proliferation of epithelial progenitors at least stage are statistically significant (Pb0.01 for all three stages up to E14.5 (De Langhe et al., 2005; Mucenski et al., 2003). examined). Thus, the lung growth defects observed in Fgf10 We therefore examined the status of canonical Wnt signaling hypomorphic embryos are not linked to general growth in Fgf10+/−; Mlc1v-LacZ+/− lungs by genetically introducing retardation. the TOPGAL allele into these mice. Previous studies indicate that canonical Wnt signaling can be detected in the epithelium Fgf10+/−;Mlc1v-LacZ+/− compound heterozygous lungs of the lung using the TOPGAL reporter mice (De Langhe et display decreased epithelial proliferation and reduction in al., 2005; Okubo and Hogan, 2004; Shu et al., 2005), where canonical Wnt activation β-galactosidase expression is a reporter for canonical Wnt signaling activation (DasGupta and Fuchs, 1999). Figs. 2E–J Analysis of PCNA positive cells as a read out for show that reduction in FGF10 signaling results in drastic proliferation was carried out in control and mutant lungs at down regulation of canonical Wnt signaling in the distal lung E14.5 and E17.5 (Figs. 2A–D). At E14.5, epithelial epithelium at E13.5 (n=3 for each genotype). β-catenin proliferation is drastically reduced in the mutant vs. control staining at E12.5 shows decreased nuclear β-catenin expres- lung (11.0±2% vs. 26.4±4.1%; n=3 for each genotype, sion in the Fgf10 hypomorph vs. control lung (Figs. 2K–N).

Fig. 3. Analysis of epithelial differentiation in Fgf10+/−;Mlc1v-LacZ+/− lungs reveals decreased expression of SP-B and TTF-1. (A–B) SP-B expression by immunohistochemistry showing a decrease in SP-B expression in newborn Fgf10+/−;Mlc1v-LacZ+/− lungs (B) compared to control (Fgf10+/−) (A). (C) The number of SP-B labeled cells per section was analyzed. The difference observed between Fgf10+/−;Mlc1v-LacZ+/− (B) and control lung (A) is significant (pb0.01). (D–E) Immunohistochemistry with TTF-1 antibody showing a decrease in TTF1 expression in newborn Fgf10+/−;Mlc1v-LacZ+/− lungs (E) compared to control (D). (F) The number of TTF-1 labelled cells per section was analyzed. The difference observed between Fgf10+/−;Mlc1v-LacZ+/− (E) and control lungs (D) is significant (pb0.03). P0 – post natal day 0. 242 S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247

Altogether, these observations suggest that FGF10 in the mesenchyme is upstream of Wnt signaling in the epithelium.

Fgf10+/−;Mlc1v-LacZ+/− compound heterozygous lungs display reduced SP-B- and TTF-1-positive cells

Immunohistochemistry was used to evaluate potential epithelial differentiation defects in Fgf10+/−;Mlc1v-LacZ+/− vs. Fgf10+/− (control) lungs at birth. Antibodies against Surfactant proteins SP-A, SP-B and SP-C as well as the general lung epithelial marker TTF-1, were used. A general reduction in the number of epithelial cells positive for these markers was detected in mutant lungs (data not shown for SP- A and SP-C). In particular, the percentile of cells positive for Surfactant Protein B was reduced by 40% in mutant vs. control (Fgf10+/−) lungs (Figs. 3A–C; 12.0±0.2% vs. 18.0± 0.2%, respectively, P b0.001). Interestingly, it has been reported that the SP-B gene mutation in human is neonatal lethal (Nogee et al., 1994). Therefore, the reduction in Surfactant Protein B expression could explain some of the respiratory failures observed at birth. However, we cannot conclude that epithelial differentiation per se is affected as we also observe a comparable reduction in the percentile of cells positive for the general epithelial marker TTF-1 in the mutant vs. control lung (Figs. 3D–F; 25.0±1.0% vs. 42.0±5.0%, respectively; n=3 for each genotype, Pb0.01). Interestingly the ratio SP-B: TTF1 in control and mutant lungs is also similar (43% vs. 48%, respectively) suggesting that the reduction in the percentile of cells positive for SP-B and TTF-1 in the mutant lung reflected a general decrease in Fig. 4. Fgf10+/−;Mlc1v-LacZ+/− lungs are not functional at birth. (A) − epithelial proliferation as observed at earlier stages (Fig. 2). Histological section of E17.5 control (Fgf10+/ ) lung depicting the canalicular +/− +/− Similar observation has been made with the ratio of SP-C: stage. (B) Section of E17.5 Fgf10 ;Mlc1v-LacZ lung. Note the larger distal airways in the mutant lung suggesting a potential defect in the septation process. TTF1 (60% vs. 55% in control vs. mutant lungs). (C) Dissected left lobe of a control (Fgf10+/−) lung at P2 (post natal day 2). (D) Dissected left lobe of Fgf10+/−;Mlc1v-LacZ+/− lung at P2. Note the extremely − − Fgf10+/ ;Mlc1v-LacZ+/ compound heterozygous lungs dilated airways of this P2 survivor. (E) H&E staining of the control lung shown − − display inhibition of secondary septa formation accompanied in panel C. (F) H&E staining of the Fgf10+/ ;Mlc1v-LacZ+/ lung shown in by respiratory failure at birth panel D. The mutant lung exhibits very dilated airways and thin mesenchyme. (G) Survival curve of Fgf10+/−;Mlc1v-LacZ+/− (n=12) showing that more than 80% of the pups die during the first 24 h. (H and I) Morphometric analysis of the +/− Histological analysis of the control and Fgf10 ; Mlc1v- lung. MLI, mean linear intercept; RAC, radio-alveolar count. Increased MLI and − LacZ+/ lungs at E13.5 and E14.5 showed no abnormalities in decreased RAC in mutant indicate dilation of the distal airways. the proximal–distal patterning of the lung epithelium. In both control and transgenic lungs, the bronchiolar proximal epithelium was columnar, whereas the distal respiratory after birth, 9 (80%) died within 24 h, 3 died within 48 h (Fig. epithelium was low cubical (data not shown). At E17.5, 4G). All of them exhibited severe gasping indicating seven out of ten lungs from Fgf10+/−;Mlc1v-LacZ+/− embryos respiratory distress. Interestingly, the 3 pups that survived displayed overly expanded distal airways with readily apparent more than 1 day after birth did not grow (data not shown) and defects in the septation process (compare Figs. 4A and B). died 48 h after birth probably as a consequence of respiratory Similar defects were found at postnatal stages (Figs. 4D–F). failure as well as limb abnormalities (Veltmaat et al., 2006 and These changes in peripheral pulmonary alveolar size were data not shown) and gut defects (Sala et al., 2006). quantified at E17.5 and P2 by morphometric measurement of mean linear intercepts (MLI) and radial-alveolar count (RAC) Fgf10+/−;Mlc1v-LacZ+/− compound heterozygous lungs (Chen et al., 2005; Figs. 4H, I). Statistically significant display reduced alveolar smooth muscle cell formation increased MLI and associated decreased RAC in mutant vs. control (Pb0.001 for the two stages examined) indicate Immunofluorescence for α-smooth muscle actin (α-SMA) dilation of the distal airways and failure to undergo secondary was used to detect alveolar smooth muscle cells at E18.5 in the septation. These structural defects in the respiratory airways respiratory airway. Fig. 5A shows the expression of α-SMA in were accompanied by early lethality. Out of 12 pups analyzed E18.5 control lungs. SMA expression was severely reduced in S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247 243 corresponding Fgf10+/−;Mlc1v-LacZ+/− lungs indicating defec- glandular stage (Fig. 6E). Formation of the microcapillaries in tive alveolar smooth muscle cell formation (n=3 for each Fgf10+/−;Mlc1v-LacZ+/− lung was impaired (Fig. 6F). Finally, genotype). In addition, in situ zymogram assays using vascular corrosion casts of the control and mutant lungs at E18.5 fluorescein isothyocyanate labeled gelatin to detect gelatinase indicate a clear simplification of the vascular tree (Fig. 6G–g′; activity (corresponding to MMP2 and MMP9) was also n=2 for control and Fgf10+/−;Mlc1v-LacZ+/−). At P2, all of performed. In this assay, the gelatin with a fluorescent tag the lungs examined exhibited large hemorrhagic areas (compare does not fluoresce unless it is cleaved and the reaction product is Figs. 6H and I). Such defects were already observed at E17.5 visualized using fluorescence microscopy. Smooth muscles in (Fig. 1H). control lung exhibit a high level of MMP activity (Fig. 5C) while a clear reduction in gelatinase activity is observed in the Decreased alveolar smooth muscle formation and reduced Fgf10+/−;Mlc1v-LacZ+/− lung (Fig. 5D). In agreement with vascular development in Fgf10+/−;Mlc1v-LacZ+/− compound reduced formation of smooth muscle cells in Fgf10+/−;Mlc1v- heterozygous lungs are associated with decreased expression LacZ+/− lung, Elastin deposition was also decreased in the of Pdgfa and Vegfa mutant lung compared to the control (data not shown). Thus the results indicate that there is a defect in the formation of alveolar We performed real-time PCR quantification of mRNA smooth muscle cells in Fgf10+/−;Mlc1v-LacZ+/− lungs. expressed in the Fgf10+/−;Mlc1v-LacZ+/− vs. Fgf10+/− lungs. Since the Fgf10+/−;Mlc1v-LacZ+/− lungs exhibited defects in Fgf10+/−;Mlc1v-LacZ+/− compound heterozygous lungs vascular development and alveolar smooth muscle cell forma- display reduced vascular development tion, expression levels of related genes were determined. Fig. 7 shows representative real-time PCR results for the relative We have investigated potential defects in the development expression of Fgf10, Vegfa, Pdgfa, and Pdgfb in Fgf10+/−; of the vascular system in Fgf10 hypomorphic lungs using Mlc1v-LacZ+/− vs. Fgf10+/− lungs. Consistent with previously light microscopy, immunofluorescence and vascular casts. reported results, we observe a 27% decrease in Fgf10 expression Figs. 6A and B show marked reduction in the number of red (Mailleux et al., 2005). Vegfa is expressed by the distal lung blood cells present in the E13.5 Fgf10+/−;Mlc1v-LacZ+/− vs. epithelium and acts on the adjacent endothelial cells, expressing Fgf10+/− (control) lungs. A general decrease in the expression VEGF-R to stimulate their proliferation (Del Moral et al., of PECAM, a marker for endothelial cells, was observed in 2006a,b).Vegfa expression was reduced by 28% in the Fgf10 E14.5 Fgf10+/−; Mlc1v-LacZ+/− vs. control lungs (n=3 for each hypomorphic lungs compared to control lungs. Pdgfa and Pdgfb genotype). Antibodies against the extracellular matrix protein are also expressed by the distal lung epithelium. These growth Laminin were also used to visualize the status of the factors act on the alveolar smooth muscle cell progenitors present microvasculature in the lung mesenchyme during the pseudo- in the mesenchyme to induce their proliferation (Bostrom et al., 1996; 2002). Pdgfa and Pdgfb expression were reduced about 60% and 16%, respectively. The expression of Hif1a and Glut1, as indicators of potential hypoxic conditions in lung tissues (Mobasheri et al., 2005), was not significantly changed between Fgf10+/− and Fgf10+/−;Mlc1v-LacZ+/− at E14.5. These results support the conclusion that changes in gene expression in the Fgf10+/−;Mlc1v-LacZ+/− lungs are solely the consequence of decreasing Fgf10 expression and not due to hypoxia.

Discussion

Fgf10−/− embryos display lung agenesis precluding functional analysis of Fgf10 during subsequent lung development. Here, we show that, in addition to its use as a reporter for Fgf10 expression, the Mlc1v-LacZ transgenic line also represents a hypomorphic allele of Fgf10. This allowed us to determine the in vivo role of Fgf10 during the later phases of lung development. Newborn Fgf10+/−;Mlc1v-LacZ+/− compound heterozygote mice exhibit severe respiratory defects and most +/+ Fig. 5. Fgf10+/−;Mlc1v-LacZ+/− lungs display reduced alveolar smooth muscle die within 24 h of birth. During embryogenesis, the Fgf10 ; +/− cell formation. (A, B) Immunohistochemistry with α-smooth muscle alpha actin Mlc1v-LacZ lungs display significant hypoplasia, associated − − (α-SMA) antibody in control (A) and Fgf10+/ ;Mlc1v-LacZ+/ P1 lung sections with dilated distal airways. Analysis of vascular development α (B). Note the drastic decrease in -SMA in the respiratory airways of the mutant shows a reduction in PECAM expression at E14.5. This lung. (C, D) Corresponding staining for Gelatinase activity (see Materials and methods for details) in control (C) and Fgf10+/−;Mlc1v-LacZ+/− lungs (D). Note reduction is associated with simplification of the vascular tree the decrease in Gelatinase activity in the mutant lung, especially around the at E18.5. E18.5 mutant lungs also display reduced expression of bronchi (arrows) and in the respiratory airway. α-SMA in the respiratory airways indicating abnormal alveolar 244 S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247

Fig. 6. Fgf10+/−;Mlc1v-LacZ+/− lungs display reduced vascular development. (A, B) E13.5 control (A) and Fgf10+/−;Mlc1v-LacZ+/− (B) lungs. Note the reduction in the presence of red blood cells in the mutant lung. (C, D) PECAM staining in control (C) and mutant (D) lung. Note the reduction in PECAM staining in the mutant. (E, F) Immunofluorescence with anti-Laminin antibodies on control (E) and mutant (F) E14.5 lungs. Note the abundant microvasculature in the mesenchyme of the control lung and the drastic reduction in development of the microvasculature in the mutant lung. DAPI: blue staining. (G) Corrosion vascular cast of control and mutant E18.5 lungs. Note the reduced complexity of the vascular tree in the mutant lung. (g, g′) Corresponding high magnification of the peripheral part of the lung casts indicated in G by boxes. (H, I) Dissected left lobe of control (H) and mutant (I) P2 lungs. Note the large hemorrhagic areas in the mutant lung supporting the previously reported vascular defects. smooth muscle cell formation. These defects are associated with Fgf10 dosage is important for proper lung development a decrease in Vegfa and Pdgfa expression, both expressed in the epithelium. The notion that appropriate levels of Fgf10 are needed for normal lung development to occur has been unclear so far. Our results indicate that the lungs of Fgf10+/− (control) mice are phenotypically normal. However, if Fgf10 levels decrease by 30% compared to heterozygous levels, distal lung hypoplasia occurs. This is a likely consequence of significant reduction in epithelial proliferation in the Fgf10 hypomorph vs. control lung. Evidence is accumulating that Fgf10 dosage is critical for proper development of certain organs while others remain unaffected. The importance of Fgf10 dosage for the development of salivary gland and lacrimal gland in humans has been shown (Entesarian et al., 2005). Individuals with autosomal dominant aplasia of lacrimal and salivary glands (ALSG) exhibit hypoplastic or missing parotid and submandibular glands. ALSG was mapped to 5p13.2–5q13.1, which includes the FGF10 gene. Subsequently, all family members with ALSG were found to be heterozygous for Fgf10. Complementary studies in adult Fgf10+/− mice revealed Fig. 7. Decreased alveolar smooth muscle formation and reduced vascular that Fgf10 heterozygotes have no parotid and smaller subman- +/− +/− development in Fgf10 ;Mlc1v-LacZ compound heterozygous lungs are dibular glands (Jaskoll et al., 2005). Our recent results further associated with decreased expression of Pdgfa and Vegfa. Real-time PCR was used to quantify and compare the expression of key genes in Fgf10+/−;Mlc1v- demonstrate that the morphogenesis of the mammary glands and LacZ+/− vs. Fgf10+/− lungs at E14.5. Representative real-time PCR data are distal region of the colon are also Fgf10 dose-dependent presented as ratio of gene expression in mutant vs. control lung. (Veltmaat et al., 2006; Sala et al., 2006). S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247 245

The role of FGF10 during the terminal maturation of lung zymography and is associated with diminished SMA expression and Elastin deposition. These data suggest that reduced Our results show that Fgf10+/−;Mlc1v-LacZ+/− pups exhibit Fgf10 expression disturbs the formation of the smooth muscle severe lung hypoplasia as a result of reduced Fgf10 expression. cells in pre-alveolar structures, possibly reflecting hypoplasia The Fgf10+/−;Mlc1v-LacZ+/− lungs displayed decreased num- of the alveolar SMC progenitors. This conclusion is supported bers of SP-A, SP-B and SP-C expressing cells at birth, by our observation that Pdgfa expression is strongly decreased suggesting that the development of the type-II alveolar cell in Fgf10+/−;Mlc1v-LacZ+/− lungs. Interestingly, Pdgfa has lineage may be affected. However, as the ratio SP-B:TTF1 also been shown to be downstream of canonical Wnt signaling (TTF1 being a general marker of lung epithelial cells) between in the lung epithelium (De Langhe et al., 2005). In addition, β- Fgf10+/− and Fgf10+/−;Mlc1v-LacZ+/− lung is similar, it is catenin has been shown to be a downstream target of FGF10 in unlikely that the decrease in surfactant protein (SP)-positive the lung epithelium (Lu et al., 2005). This is in harmony with cells reflects a defect in epithelial differentiation. Our results the results of our recent Affymetrix arrays showing a 13% suggest that the decrease in epithelial cell proliferation during decrease in β-catenin expression in Fgf10 hypomorph vs. the pseudoglandular stage is the underlying cause of the control lung at E14.5 (data not shown). However, it is unlikely reduction in SP-positive cells. that decreased transcription of β-catenin per se is enough to Many in vitro studies imply FGFs as signals of proliferation explain the reduction in Wnt signaling observed in the Fgf10 and differentiation (for reviews see Cardoso, 2001; Cardoso and hypomorphic lungs (Fig. 2). Our results show indeed a Lu, 2006; Warburton et al., 2000). In vivo, the loss of function decrease in nuclear β-catenin localization in the epithelium of studies of Fgfr2b or Fgf10 show clearly that this signaling Fgf10 hypomorph vs. control lungs (Fig. 2). It has recently pathway is vital in the development of the bronchial tree structure been shown that the well-known PI3K/AKT pathway is (Arman et al., 1999; De Moerlooze et al., 2000; Min et al., 1998; capable, via the phosphorylation of β-catenin on serine 522, to Sekine et al., 1999). Nevertheless, the phenotypes of the null increase the amount of nuclear β-catenin (He et al., 2007). It is mutants make it impossible to establish the functional role of therefore likely that in the lung epithelium, FGF10 controls β- FGF10 and FGFR2b in late stages of pulmonary development. In catenin signaling in this way rather than at the level of the the case of Fgfr2b, the inducible over-expression of the soluble transcription of the β-catenin gene. More work will have to be dominant negative FGFR2b receptor from the end of the done to determine the exact mechanism of action of FGF10 on pseudoglandular phase (E14.5) disturbs alveogenesis after the Wnt/β-catenin signaling. Our result that TOPGAL birth. By contrast, a similar inhibition of FGFR2b signals after expression is decreased in Fgf10+/−;Mlc1v-LacZ+/− lungs, birth does not lead to a defect in alveolar formation suggesting therefore strongly suggests that FGF10, upstream of β-catenin that FGFR2b signaling is not needed for lung homeostasis signaling, controls Pdgfa expression in the lung epithelium. (Hokuto et al., 2003). The authors reported that FGFR2b signaling is indeed critical during the early pseudoglandular FGF10 is a key regulator of epithelial morphogenesis and stage. Decrease in FGFR2b signaling during this early develop- vascular development mental phase impacts negatively alveogenesis. Interestingly, other FGFR2b ligands (FGF1, 3, 7) are expressed from E13.5 Decreased PECAM and Laminin expression at E14.5 in onwards in lung, either in the epithelium or the mesenchyme Fgf10 hypomorphic lungs suggest that the simplified vascular (Bellusci et al., 1997; Cardoso, 2001; Lebeche et al., 1999). tree observed later on, at E18.5, is likely the consequence of There are therefore many FGFR2b ligands that could potentially abnormal vascular development starting early during the compensate for the loss of Fgf10. However, Fgf10 hypomorphic pseudoglandular stage. Interestingly, we have demonstrated mice have alveolar defects, as the likely consequence of events that lung development during the pseudoglandular stage is taking place during early embryonic development, when Fgf10 under the unique control of Fgf10. Our results therefore suggest is the only functional FGFR2b ligand expressed in the lung. Our a link between mesenchymal FGF10, which acts directly on the results therefore demonstrate that FGF10 plays a unique function epithelium and the development of the vascular system. This in the E9.5–E13.5 pseudoglandular period specifically in the observation has important implications as epithelial morpho- amplification of pulmonary epithelial progenitors. genesis, mostly mediated by FGF10, has to be tightly controlled with vascular development to obtain a functional lung at birth. FGF10 indirectly controls the formation of the alveolar smooth Our results indicate that there is a 28% decrease in Vegfa muscle cells expression in the Fgf10 hypomorph vs. control lung. Interest- ingly, a similar decrease in TTF1 expression is observed in the A similar emphysematous-like, non-inflammatory lung mutant lungs indicating that the decrease in Vegfa is due to the phenotype and postnatal death has been described for the Elas- reduction in the number of epithelial cells and not because tin and Mmp-2 null mice (Kheradmand et al., 2002; Wendel et FGF10 controls directly, at the transcriptional level, Vegfa al., 2000). In 15% of Mmp-2−/− newborns, a syndrome of expression in the epithelium. acute respiratory distress is associated with improper deposi- In conclusion, our results indicate that FGF10 plays a crucial tion of elastin fibers (Kheradmand et al., 2002). Interestingly, a role throughout the first half of the pseudoglandular stage in specific decrease in gelatinase activity at birth in Fgf10+/−; maintaining the activation of the Wnt canonical pathway in Mlc1v-LacZ+/− lung mesenchyme was revealed by in situ epithelial progenitor cells and regulating their proliferation. In 246 S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247 addition, we have demonstrated that FGF10 is essential in Herlenius, M., Dahl, N., 2005. Mutations in the gene encoding fibroblast coordinating alveolar smooth muscle cell formation and growth factor 10 are associated with aplasia of lacrimal and salivary glands. Nat. Genet. 37, 125–127. vascular development. He, X.C., Yin, T., Grindley, J.C., Tian, Q., Sato, T., Tao, W.A., Dirisina, R., Porter-Westpfahl, K.S., Hembree, M., Johnson, T., Wiedemann, L.M., Acknowledgments Barrett, T.A., Hood, L., Wu, H., Li, L., 2007. PTEN-deficient intestinal stem cells initiate intestinal polyposis. Nat. Genet. 39, 189–198. This work was funded by ALA and an NIH RO1 HL074832 Hokuto, I., Perl, A.K., Whitsett, J.A., 2003. Prenatal, but not postnatal, inhibition of fibroblast growth factor receptor signaling causes emphysema. (to SB), HL60231, 75773, 44060, 44977 (to DW), HL056590 J. Biol. Chem. 278, 415–421. and 073471 (to PM), CBCRP and CHLA institutional award (to Jaskoll, T., Abichaker, G., Witcher, D., Sala, F.G., Bellusci, S., Hajihosseini, JV); ARC and FRM (to AA), CHLA institutional award (to M.K., Melnick, M., 2005. FGF10/FGFR2b signaling plays essential roles PdM), ALA and CHLA institutional award (to SDL). We would during in vivo embryonic submandibular salivary gland morphogenesis. like to thank Dennis Mock for his technical help with the BMC Dev. Biol. 5, 11. Kelly, R., Alonso, S., Tajbakhsh, S., Cossu, G., Buckingham, M., 1995. Myosin analysis of the Affymetrix arrays. light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice. J. Cell Biol. 129, 383–396. Kelly, R.G., Brown, N.A., Buckingham, M.E., 2001. The arterial pole of the References mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev. Cell 1, 435–440. Arman, E., Haffner-Krausz, R., Gorivodsky, M., Lonai, P., 1999. Fgfr2 is Kheradmand, F., Rishi, K., Werb, Z., 2002. Signaling through the EGF receptor required for limb outgrowth and lung-branching morphogenesis. Proc. Natl. controls lung morphogenesis in part by regulating MT1–MMP-mediated Acad. Sci. U. S. A. 96, 11895–11899. activation of gelatinase A/MMP2. J. Cell Sci. 115, 839–848. Bellusci, S., Grindley, J., Emoto, H., Itoh, N., Hogan, B.L., 1997. Fibroblast Lebeche, D., Malpel, S., Cardoso, W.V., 1999. Fibroblast growth factor growth factor 10 (FGF10) and branching morphogenesis in the embryonic interactions in the developing lung. Mech. Dev. 86, 125–136. mouse lung. Development 124, 4867–4878. Lu, J., Izvolsky, K.I., Qian, J., Cardoso, W.V., 2005. Identification of FGF10 Bostrom, H., Willetts, K., Pekny, M., Leveen, P., Lindahl, P., Hedstrand, H., targets in the embryonic lung epithelium during bud morphogenesis. J. Biol. Pekna, M., Hellstrom, M., Gebre-Medhin, S., Schalling, M., Nilsson, M., Chem. 280, 4834–4841. Kurland, S., Tornell, J., Heath, J.K., Betsholtz, C., 1996. PDGF-A signaling Mailleux, A.A., Tefft, D., Ndiaye, D., Itoh, N., Thiery, J.P., Warburton, D., is a critical event in lung alveolar myofibroblast development and Bellusci, S., 2001. Evidence that SPROUTY2 functions as an inhibitor of alveogenesis. Cell 85, 863–873. mouse embryonic lung growth and morphogenesis. Mech. Dev. 102, Bostrom, H., Gritli-Linde, A., Betsholtz, C., 2002. PDGF-A/PDGF alpha- 81–94. receptor signaling is required for lung growth and the formation of alveoli Mailleux, A.A., Spencer-Dene, B., Dillon, C., Ndiaye, D., Savona-Baron, C., but not for early lung branching morphogenesis. Dev. Dyn. 223, 155–162. Itoh, N., Kato, S., Dickson, C., Thiery, J.P., Bellusci, S., 2002. Role of Burns, R.C., Fairbanks, T.J., Sala, F., De Langhe, S., Mailleux, A., Thiery, J.P., FGF10/FGFR2b signaling during mammary gland development in the Dickson, C., Itoh, N., Warburton, D., Anderson, K.D., Bellusci, S., 2004. mouse embryo. Development 129, 53–60. Requirement for fibroblast growth factor 10 or fibroblast growth factor Mailleux, A.A., Kelly, R., Veltmaat, J.M., De Langhe, S.P., Zaffran, S., Thiery, receptor 2-IIIb signaling for cecal development in mouse. Dev. Biol. 265, J.P., Bellusci, S., 2005. Fgf10 expression identifies parabronchial smooth 61–74. muscle cell progenitors and is required for their entry into the smooth Cardoso, W.V., 2001. Molecular regulation of lung development. Annu. Rev. muscle cell lineage. Development 132, 2157–2166. Physiol. 63, 471–494. Min, H., Danilenko, D.M., Scully, S.A., Bolon, B., Ring, B.D., Tarpley, J.E., Cardoso, W.V., Lu, J., 2006. Regulation of early lung morphogenesis: questions, DeRose, M., Simonet, W.S., 1998. Fgf-10 is required for both limb and lung facts and controversies. Development 133, 1611–1624. development and exhibits striking functional similarity to Drosophila Chen, H., Sun, J., Buckley, S., Chen, C., Warburton, D., Wang, X.F., Shi, W., branchless. Genes Dev. 12, 3156–3161. 2005. Abnormal mouse lung alveolarization caused by Smad3 deficiency is Mobasheri, A., Richardson, S., Mobasheri, R., Shakibaei, M., Hoyland, J.A., a developmental antecedent of centrilobular emphysema. Am. J. Physiol., 2005. Hypoxia inducible factor-1 and facilitative glucose transporters Lung Cell. Mol. Physiol. 288, L683. GLUT1 and GLUT3: putative molecular components of the oxygen and DasGupta, R., Fuchs, E., 1999. Multiple roles for activated LEF/TCF glucose sensing apparatus in articular chondrocytes. Histol. Histopathol. 20, transcription complexes during hair follicle development and differentiation. 1327–1338. Development 126, 4557–4568. Mucenski, M.L., Wert, S.E., Nation, J.M., Loudy, D.E., Huelsken, J., De Langhe, S.P., Sala, F.G., Del Moral, P.M., Fairbanks, T.J., Yamada, K.M., Birchmeier, W., Morrisey, E.E., Whitsett, J.A., 2003. beta-Catenin is Warburton, D., Burns, R.C., Bellusci, S., 2005. Dickkopf-1 (DKK1) reveals required for specification of proximal/distal cell fate during lung that fibronectin is a major target of Wnt signaling in branching morphogenesis. J. Biol. Chem. 278, 40231–40238. morphogenesis of the mouse embryonic lung. Dev. Biol. 277, 316–331. Nogee, L.M., Garnier, G., Dietz, H.C., Singer, L., Murphy, A.M., deMello, D.E., De Moerlooze, L., Spencer-Dene, B., Revest, J., Hajihosseini, M., Rosewell, I., Colten, H.R., 1994. A mutation in the surfactant protein B gene responsible Dickson, C., 2000. An important role for the IIIb isoform of fibroblast for fatal neonatal respiratory disease in multiple kindreds. J. Clin. Invest. 93, growth factor receptor 2 (FGFR2) in mesenchymal–epithelial signalling 1860–1863. during mouse organogenesis. Development 127, 483–492. Okubo, T., Hogan, B.L., 2004. Hyperactive Wnt signaling changes the Del Moral, P.M., De Langhe, S.P., Sala, F.G., Veltmaat, J.M., Tefft, D., Wang, developmental potential of embryonic lung endoderm. J. Biol. 3, 11. K., Warburton, D., Bellusci, S., 2006a. Differential role of FGF9 on Park, W.Y., Miranda, B., Lebeche, D., Hashimoto, G., Cardoso, W.V., 1998. epithelium and mesenchyme in mouse embryonic lung. Dev. Biol. 293, FGF-10 is a chemotactic factor for distal epithelial buds during lung 77–89. development. Dev. Biol. 201, 125–134. Del Moral, P.M., Sala, F.G., Tefft, D., Shi, W., Keshet, E., Bellusci, S., Sala, F.G., Curtis, J.L., Veltmaat, J.M., Del Moral, P.M., Le, L.T., Fairbanks, T. Warburton, D., 2006b. VEGF-A signaling through Flk-1 is a critical J., Warburton, D., Ford, H., Wang, K., Burns, C.R., Bellusci, S., 2006. facilitator of early embryonic lung epithelial to endothelial crosstalk and Fibroblast Growth Factor 10 is required for survival and proliferation but not branching morphogenesis. Dev. Biol. 290, 177–188. differentiation of intestinal epithelial progenitor cells during murine colon Entesarian, M., Matsson, H., Klar, J., Bergendal, B., Olson, L., Arakaki, R., development. Dev. Biol. 299, 373–385. Hayashi, Y., Ohuchi, H., Falahat, B., Bolstad, A.I., Jonsson, R., Wahren- Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247 247

Yagishita, N., Matsui, D., Koga, Y., Itoh, N., Kato, S., 1999. Fgf10 is the induction and patterning of mammary epithelium along the embryonic essential for limb and lung formation. Nat. Genet. 21, 138–141. axes. Development 133, 2325–2335. Shu, W., Guttentag, S., Wang, Z., Andl, T., Ballard, P., Lu, M.M., Piccolo, S., Warburton, D., Schwarz, M., Tefft, D., Flores-Delgado, G., Anderson, K.D., Birchmeier, W., Whitsett, J.A., Millar, S.E., Morrisey, E.E., 2005. Wnt/ Cardoso, W.V., 2000. The molecular basis of lung morphogenesis. Mech. beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling Dev. 92, 55–81. to regulate proximal–distal patterning in the lung. Dev. Biol. 283, Weaver, M., Dunn, N.R., Hogan, B.L., 2000. Bmp4 and Fgf10 play opposing 226–239. roles during lung bud morphogenesis. Development 127, 2695–2704. Veltmaat, J.M., Relaix, F., Le, L.T., Kratochwil, K., Sala, F.G., van Veelen, W., Wendel, D.P., Taylor, D.G., Albertine, K.H., Keating, M.T., Li, D.Y., 2000. Rice, R., Spencer-Dene, B., Mailleux, A.A., Rice, D.P., Thiery, J.P., Bellusci, Impaired distal airway development in mice lacking elastin. Am. J. Respir. S., 2006. Gli3-mediated somitic Fgf10 expression gradients are required for Cell Mol. Biol. 23, 320–326.