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; 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.04.033 238 S.K. Ramasamy et al. / Developmental Biology 307 (2007) 237–247 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.