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. Author manuscript; available in PMC: 2013 Jan 6.
Published in final edited form as: Dev Dyn. 2009 Aug;238(8):1999–2013. doi: 10.1002/dvdy.22032

Conditional Gene Inactivation Reveals Roles for Fgf10 and Fgfr2 in Establishing a Normal Pattern of Epithelial Branching in the Mouse Lung

Lisa L Abler 1, Suzanne L Mansour 2, Xin Sun 1,*
PMCID: PMC3538083  NIHMSID: NIHMS153538  PMID: 19618463

Abstract

Fibroblast growth factor 10 (FGF10) signaling through FGF receptor 2 (FGFR2) is required for lung initiation. While studies indicate that Fgf10 and Fgfr2 are also important at later stages of lung development, their roles in early branching events remain unclear. We addressed this question through conditional inactivation of both genes in mouse subsequent to lung initiation. Inactivation of Fgf10 in lung mesenchyme resulted in smaller lobes with a reduced number of branches. Inactivation of Fgfr2 in lung epithelium resulted in disruption of lobes and small epithelial outgrowths that arose arbitrarily along the main bronchi. In both mutants, there was an increase in cell death. Also, the expression patterns of key signaling molecules implicated in branching morphogenesis were altered and a proximal lung marker was expanded distally. Our results indicate that both Fgf10 and Fgfr2 are required for a normal branching program and for proper proximal-distal patterning of the lung.

Keywords: Fgf10, Fgfr2, lung, development, branching morphogenesis, cell survival, proximal-distal patterning

Introduction

The lung is composed of a complex network of epithelial branches, which is critical for normal organ function. This network arises from three modes of branch formation used iteratively in relatively simple sequences (Metzger et al., 2008). Evidence of such stereotypical patterning implies that there is strict genetic control of branching in the lung. Branch formation and patterning are directed by reciprocal signaling interactions between epithelial and mesenchymal cell populations (Cardoso and Lu, 2006). These interactions are mediated by a number of signaling molecules that, along with their receptors, are expressed dynamically in lung epithelium, mesenchyme or both.

Fibroblast growth factor (FGF) signaling has been shown to mediate epithelial-mesenchymal interactions during lung development. Six FGFs are expressed in the lung but, of these, only Fgf10 has been shown to be essential for lung formation, a process that initiates at embryonic day (E) 9.5 (Warburton et al., 2005). Several lines of evidence indicate that Fgf10 acts as a chemoattractant for the lung epithelium. Fgf10 is expressed in discretely localized sites in the mesenchyme from the earliest stages of lung formation, a pattern that shifts dynamically to sites of prospective bud formation throughout development (Bellusci et al., 1997). In vitro studies show that mesenchyme-free distal epithelial tips cultured in media containing recombinant FGF10 expand to form a cyst-like structure and then bud (Bellusci et al., 1997). Additionally, denuded epithelial tips proliferate and migrate toward an FGF10-loaded bead (Weaver et al., 2000).

Genetic evidence likewise indicates that FGF signaling plays a key role in early lung development. Fgf10 knockout mice develop a trachea but fail to form a lung, demonstrating that Fgf10 is required for lung initiation (Min et al., 1998; Sekine et al., 1999). Analysis of FGF receptor (FGFR) mutants reveals a similar requirement. Fgfr2 null mice die shortly after implantation, but fusion chimeras carrying a null allele of Fgfr2 surpass this early requirement and survive until birth (Arman et al., 1998; Arman et al., 1999; De Moerlooze et al., 2000). These mutants exhibit lung agenesis, as do mice carrying a null allele of Fgfr2b, an alternatively spliced isoform of Fgfr2 expressed only in the epithelium (De Moerlooze et al., 2000). Both the chimera and the Fgfr2b mutants exhibit lung phenotypes resembling that seen in Fgf10 null mice. These data, when considered along with evidence from mitogenic assays demonstrating that FGF10 binds with high affinity and specificity to FGFR2b (Zhang et al., 2006), indicate that an FGF10-FGFR2 pathway is required for lung initiation.

Experimental evidence suggests that, in addition to their critical roles during lung initiation, Fgf10 and Fgfr2 continue to function later in lung development. A recent study showed that insertion of a lacZ cassette into the regulatory region of Fgf10 creates an Fgf10 hypomorphic mutant (Kelly et al., 2001; Mailleux et al., 2005). These mice have smaller lungs at or near birth in which epithelial branching is decreased (Ramasamy et al., 2007). Transgenic expression of Fgf10 in the lung epithelium disrupts branching morphogenesis perinatally and results in the formation of lung tumors (Clark et al., 2001). Similarly, when a soluble dominant negative form of Fgfr2 is transgenically expressed in the lung epithelium, fewer branches are observed perinatally while postnatal lungs exhibit emphysema (Peters et al., 1994; Hokuto et al., 2003). These results describe a role for Fgf10 and Fgfr2 in epithelial branch formation at later stages of lung development.

Collectively, the aforementioned studies focus primarily on later developmental events such as epithelial and mesenchymal cell differentiation and number of terminal epithelial branches. Though at least one group mentions reduced branching at E12.5 (Ramasamy et al., 2007), none of these studies directly addresses the roles of Fgf10 and Fgfr2 in branching morphogenesis subsequent to lung initiation. In this study, we conditionally inactivated each of these genes to investigate their functions in epithelial branching at early stages of lung development. We found that ramification of epithelial branches is reduced in both cases, to varying degrees. We characterized observed changes by examining several aspects of lung development. We show that cell survival declines in these mutants. We examined the effects that inactivation of Fgf10 and Fgfr2 have on other signaling pathways essential for lung development. The expression of several key molecules, including bone morphogenetic protein 4 (Bmp4), sonic hedgehog (Shh) and Fgf10 itself, is altered as a result of decreased FGF10-FGFR2 signaling. Finally, we show that proximal-distal (P-D) patterning of the lung is perturbed in these mutants.

Results

Inactivation of Fgf10 in lung mesenchyme and Fgfr2 in lung epithelium using conditional alleles

To define the roles of FGF10-FGFR2 signaling in lung branching morphogenesis using a genetic approach, we inactivated either the ligand or the receptor in the developing mouse lung using existing conditional alleles. As the findings from these two mutants reinforce each other, we will present the results in parallel. To inactivate Fgf10 during early stages of branching morphogenesis, we obtained a conditional allele of Fgf10 (Fgf10flox) in which loxP sites were inserted flanking the second exon (flox). Cre-mediated deletion of this exon yields a null allele of Fgf10. Similar to other Fgf10 null alleles, pups globally homozygous for the exon 2 deletion lack lungs and die at birth (X. Wang and S. Mansour, unpublished). We used the Dermo1cre line to inactivate Fgf10 throughout the lung mesenchyme (Yu et al., 2003). For a detailed analysis of Dermo1cre activity in the lung during early stages of development, we mated mice carrying this allele to a ROSA26 reporter (R26R) line that reports Cre activity through expression of lacZ (Soriano, 1999). In the lung mesenchyme of Dermo1cre/+;R26R (hereafter Dermo1-cre;R26R) samples at E10.5, recombination was biased, with more activity in rostral than in caudal regions (Fig. 1A). However, by E12, activity was evenly distributed throughout the lung (Fig. 1B). These results indicate that Dermo1cre activity is widespread throughout the lung mesenchyme shortly after lung initiation.

Figure 1.

Figure 1

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Dermo1cre/+ activity and Fgf10 inactivation.

(A,B) Cre activity of the Dermo1cre/+ allele as assayed by β–gal staining of Dermo1- cre;R26R embryos at stages indicated. Dotted line in A frames caudal periphery of lung. Dotted lines in B outline each of the lobes: L, left; Ro, rostral; Me, medial; Ac, accessory and Ca, caudal. (C) Amount of intact Fgf10 transcript remaining per lobe of Dermo1-cre;Fgf10 mutant lung as determined by qRT-PCR. Full length (exon 2-containing) Fgf10 transcript was undetectable in the mutant rostral lobe. (D-G) FGF signaling as assayed by RNA in situ hybridization using a Spry2 probe as a readout of FGF signaling at stages indicated. Arrowheads indicate comparable regions of the lung to highlight differences in Spry2 expression. Arrows indicate normal position of the rostral lobe (solid arrow) to illustrate its absence in the mutant (open arrow). Ventral views of lungs shown in all figures unless otherwise indicated.

To examine the timing and extent of Fgf10 inactivation in the lung by Dermo1cre, we performed quantitative RT-PCR (qRT-PCR) analysis using primers that amplify Fgf10 exon 2, which is deleted in mesenchymal cells expressing Dermo1cre. Individual lobes of Dermo1cre/+;Fgf10null/flox (hereafter Dermo1-cre;Fgf10) mutant lungs were collected at E12.5 and compared to control lobes. Full length (exon 2-containing) Fgf10 transcript was undetectable in the rostral lobe and was reduced in the rest of the mutant lobes, ranging from a 15% reduction in the caudal lobe to a 60% reduction in the accessory lobe (Fig. 1C). Slightly lower levels of transcript were detected at E13.5 (data not shown). These data suggest that varying amounts of intact Fgf10 transcript remain in different lobes of the lung, likely reflecting the regional differences in recombination efficiency observed early in mutant lung development.

To confirm qRT-PCR data and determine where remaining FGF signaling is active, we performed RNA in situ hybridization analysis using a sprouty2 (Spry2) probe as readout of FGF signaling because Spry expression is induced by FGF family members (Minowada et al., 1999). In control lung samples at E11.25, Spry2 is expressed at high levels in the distal tips of epithelial buds. In Dermo1-cre;Fgf10 mutants at E11.25 and E12.5, Spry2 was expressed at progressively decreasing intensities (Fig.1D-G). This decrease was more pronounced in rostral than in caudal regions. These results indicate that low levels of FGF signaling remain in Dermo1-cre;Fgf10 mutant lungs and inactivation is more efficient in rostral than in caudal mesenchyme.

We next inactivated Fgfr2 in the lung using a conditional allele (Fgfr2flox). In this allele, loxP sites flank exons encoding part of the ligand binding third immunoglobulin-like (Ig) motif and the transmembrane domain. Cre-mediated deletion of this flanked region yields a null allele (Yu et al., 2003). In order to inactivate Fgfr2 specifically in the lung epithelium, we employed a surfactant protein C-cre (Sftpc-cre) line in which Cre recombinase is expressed under the control of Sftpc promoter elements (Okubo et al., 2005). For a detailed analysis of Sftpc-cre activity in the lung, we crossed this line to the R26R reporter line (Soriano, 1999). We first detected β–galactosidase (β-gal) activity in a few isolated cells in the lung epithelium of Sftpc-cre;R26R mice at E9.75 (data not shown). By E10.5, one day after lung initiation, β-gal activity was detected throughout the lung epithelium. At this stage, however, activity appeared to be mosaic with less staining in the caudal part of the primary buds, particularly in the right lobe (Fig. 2A,B). Cre-mediated reporter recombination was mostly complete by E12.5, though a limited number of unrecombined cells were scattered throughout the right caudal lobe of several samples (n = 7/12) (Fig. 2C,D). These results suggest that the Sftpc-cre transgene confers widespread Cre-mediated recombination throughout the lung epithelium shortly after lung initiation.

Figure 2.

Figure 2

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Sftpc-cre activity and Fgfr2 inactivation.

(A-D) Cre activity of the Sftpc-cre transgene as assayed by β–gal staining of Sftpc-cre;R26R embryos at stages indicated. B is a lateral view of the right side of the lung shown in A. Dotted lines in A, B frame caudal periphery of lung. D is a magnified view of the bracketed region indicated in panel C. Uneven β-gal signal indicates variations in the efficiency of recombination. (E-I) An RNA in situ hybridization probe targeted to floxed exons of Fgfr2 (Fgfr2 exon8,10) was used to detect intact receptor in lungs of indicated genotypes and stages. (J-N) FGF signaling as assayed using a Spry2 in situ probe. Arrowheads in J,K indicate comparable regions of the lung to illustrate differences in Spry2 expression. I, N are magnified views of bracketed regions in H, M, respectively. Arrowheads indicate regions of residual Fgfr2 exon8,10 (I) or Spry2 (N) expression. Esophagus is included in this figure and others as an internal standard for equivalence of staining between control and mutant samples.

To address the timing and extent of Fgfr2 inactivation by Sftpc-cre, we performed RNA in situ hybridization analysis using a probe that hybridizes to deleted exons of Fgfr2 (Fgfr2 exon 8,10). At E10.5, Fgfr2 exon 8,10 mRNA was detected in both control and Sftpc-cre;Fgfr2flox/flox (hereafter, Sftpc-cre;Fgfr2) mutant lungs (data not shown). At E11.25, Fgfr2 exon 8,10 mRNA was detected throughout the epithelium of control samples and was present, though at reduced levels, in mutant samples (Fig. 2E,F). By E12.5, Fgfr2 exon 8,10 mRNA was not detected in the proximal epithelium of mutant lungs but remained expressed at low levels in isolated patches of distal epithelium, especially in the caudal regions of both lobes (Fig. 2G-I). These results indicate that Fgfr2 is largely inactivated in the lung epithelium by E12.5.

To determine whether residual Fgfr2 mRNA in Sftpc-cre;Fgfr2 lungs can transduce an FGF signal, we assayed for FGF signaling using a Spry2 in situ probe. In mutant samples, though distal upregulation was maintained in epithelial bud tips at E11.25, the domains of Spry2 expression appeared smaller (Fig. 2J,K). At E12.5, Spry2 expression remained in only a few distal epithelial cells located in the caudal regions of both lobes (Fig. 2L-N). Since Spry2 expression closely correlates with residual Fgfr2 exon 8,10 expression, it appears that those areas of residual signaling coincide with the presence of intact Fgfr2 transcripts. Compared to Dermo1-cre;Fgf10 mutants, Spry2 appears to be expressed in fewer, scattered epithelial cells in Sftpc-cre;Fgfr2 lungs, suggesting that FGF signaling is more severely disrupted in the receptor mutant than in the ligand mutant.

Inactivation of Fgf10 or Fgfr2 results in striking defects in branching pattern

In order to visualize the branching pattern, in situ analysis was used to outline the epithelium of control and Dermo1-cre;Fgf10 or Sftpc-cre;Fgfr2 mutant lungs. We utilized a second Fgfr2 probe that hybridizes to a portion of the Fgfr2 mRNA still produced in the Sftpc-cre;Fgfr2 mutant, a region which normally encodes a tyrosine kinase domain (hereafter referred to as Fgfr2 TK) (Peters et al., 1992). To ensure that observed phenotypic differences were not compounded by variations in developmental stage, mutant lungs were compared to those from somite-matched littermates throughout this study. In wild type mouse lungs at E11.25, the epithelial branches that establish the four lobes of the right lung are readily visible. At this stage, we often were able to identify Dermo1-cre;Fgf10 mutants due to reduction or absence of the nascent rostral lobe (absent in n = 32/45 samples) (Fig. 3A,B). At E12.5, mutant lungs were smaller than their control littermates. The rostral lobe was the most severely affected of the lobes and was absent in a majority of samples (n = 35/56) (Fig. 3C,D). The remaining lobes were present and branching did occur within them, but they tended to be smaller than control lobes. The medial and accessory lobes, in particular, were quite reduced in size and often misshapen. All lobes exhibited reduced branching following outgrowth of the initial branch that established the lobe. Because any branches that did form by E12.5 appeared to be morphologically normal, we followed the Dermo1-cre;Fgf10 mutant phenotype out to E17.5 in whole-mount (Fig. 3E). At this stage, mutant lungs were severely hypoplastic and often exhibited hemorrhaging. Though it was evident that branching morphogenesis had continued, i.e. the branches had not arrested, lobe shape was highly abnormal in these mutants. For example, mutant lobes were smaller and much flatter than control lobes. These differences cannot be attributed to delayed branching, as mutant lungs at E17.5 do not resemble control lungs at earlier stages of development. These results indicate that Fgf10 is required for establishing a normal branch pattern.

Figure 3.

Figure 3

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Morphology of Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutant lungs.

(A-E) Epithelial branching pattern of Dermo1-cre;Fgf10 lungs. (A-D) An Fgfr2 TK probe was used to outline the epithelium in lungs of indicated genotypes and stages. Solid arrows in A, C and dotted line in C indicate normal position of the rostral lobe and open arrows in B and D indicate absence of the rostral lobe in the mutant. (E) Samples shown in whole-mount at E17.5. (F-L) Epithelial branching pattern of Sftpc-cre;Fgfr2 lungs as outlined by Fgfr2 TK in situ at stages indicated. Arrow in K indicates a dilated sac. Bracketed region in K is magnified in inset; arrowheads indicate small nodules. L is a magnified view of the right lung shown in K; arrow indicates outgrowth of lobe mesenchyme without outgrowth of epithelium.

Sftpc-cre;Fgfr2 mutants also exhibited defects in epithelial branching. At E10.5, the appearance of the outgrowing bronchi was the same in both control and Sftpc-cre;Fgfr2 mutant lungs, indicating that lung initiation occurred normally in these mutants (Fig. 3F,G). However, by E11.25, a branching defect was readily apparent in mutant lungs. Branches forming in the right lung, though approximately in the correct locations, were smaller and had a bumpy, irregular morphology, whereas some samples lacked definitive lobar branches altogether (Fig. 3H,I). The overall size of the mutant lungs was comparable to controls at this stage. By E12.5, irregular outgrowths had arisen randomly along the entire length of both main bronchi of the mutant lungs, though these outgrowths tended to be more concentrated caudally. The morphology of these outgrowths varied widely, from small nodules to dilated sacs (Fig. 3J,K). None of the outgrowths observed appeared to undergo further branching events at the stages examined. Sftpc-cre;Fgfr2 mutants were noticeably smaller than their littermate controls by E12.5. These phenotypes indicate that epithelial Fgfr2, like mesenchymal Fgf10, is required for normal branch formation.

In both Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants, we frequently observed mesenchymal protrusions without an accompanying epithelial branch (Fig. 3L, 5D,L). These protrusions were observed only in the right lung in positions that correspond to lobes of the lung. For example, in Dermo1-cre;Fgf10 mutants that developed a rudimentary rostral lobe, that lobe consisted only of a small piece of mesenchymal tissue and lacked observable epithelial outgrowth from the main bronchus (Fig. 5D). A similar mesenchymal protrusion often was seen in Sftpc-cre;Fgfr2 mutants in the approximate position of the medial lobe (Fig. 3L, 5L). These observations suggest that mesenchymal outgrowth can occur independently of epithelial branching.

Figure 5.

Figure 5

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Shh and Ptch1 expression in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutant lungs.

(A-P) Gene expression in lungs of indicated genotypes and stages. Arrowheads in A,B indicate comparable regions of the lung to illustrate differences in Shh expression. Bracketed regions in C-D and K-L are magnified in insets. Shh is not upregulated in the distal epithelium of the Dermo1-cre;Fgf10 mutant but remains upregulated in the Sftpc-cre;Fgfr2 mutant, albeit in smaller domains. Dotted line in D and L indicates outgrowth of lobe mesenchyme without outgrowth of epithelium. (Q-T) Gene expression in wild type lungs cultured with protein-soaked beads (dashed circles). Arrows in R, T indicate domains of upregulated expression in tissue immediately adjacent to the FGF10-soaked beads.

Ectopic cell death is observed in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants

To examine whether perturbation of FGF signaling affected cell survival in Dermo1- cre;Fgf10 or Sftpc-cre;Fgfr2 mutants, we analyzed cell death using LysoTracker staining to label apoptotic cells. In normal controls at E11.25, cell death is detected at low levels throughout the length of the trachea and in the primary bronchi just past the point of bifurcation. The pattern of epithelial cell death in most Dermo1-cre;Fgf10 mutants (n = 4/6) was comparable to normal controls, though a couple of mutants (n = 2/6) exhibited a somewhat higher intensity of cell death that extended along almost the entire length of the primary bronchi (compare Fig. 4A,B). At E12, controls exhibit moderate levels of cell death throughout the trachea, which extends into the primary bronchi of both right and left lungs. In Dermo1-cre;Fgf10 mutants, epithelial cell death was detected at increased intensity. In addition, cell death extended farther into the medial and accessory branches and there was a small area of ectopic death observed in the mesenchyme of either the vestigial rostral lobe (data not shown) or at the distal tip of the accessory lobe (n = 3/3) (compare Fig. 4C,D). Thus, Dermo1-cre;Fgf10 mutants exhibited aberrant cell death, indicating that FGF10 is essential for cell survival.

Figure 4.

Figure 4

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Cell death analysis of Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants.

(A-H) LysoTracker staining of apoptotic cells in lungs of indicated genotypes and stages. For all samples, dotted lines outline lobes of lungs and arrows indicate caudal extent of cell death in epithelium of primary bronchi. Arrowheads in C, D indicate cell death in epithelium of medial and accessory lobes. Asterisks in D, H indicate aberrant mesenchymal death. (I-M) Immunofluorescent staining on paraffin sections of lungs of indicated genotypes and stages. Cleaved Caspase 3 antibody staining labels dying cells within the trachea (I, K, M). E-Cadherin antibody staining on adjacent serial sections outlines the tracheal epithelium (J, L, N). Arrows in I, K, M indicate cell death within the tracheal epithelium.

Ectopic cell death was also observed in Sftpc-cre;Fgfr2 mutants. At E10.5, localization of cell death was comparable between normal controls and Sftpc-cre;Fgfr2 mutants (n = 3/3) (data not shown). At E11.25, while some mutants exhibited a pattern of epithelial cell death similar to controls, apoptosis was detected at higher intensity and was expanded caudally in the bronchial epithelium of other mutants (n = 3/7) (compare Fig. 4E,F). By E12.5, Sftpc-cre;Fgfr2 mutants revealed widespread cell death throughout the entire epithelium. Additionally, cell death was observed in the peripheral mesenchyme of mutant lungs, especially in caudal regions (n = 2/2) (compare Fig. 4G,H). As Fgfr2 was inactivated specifically in the lung epithelium, this mesenchymal death is secondary to a defect in the epithelium. These data suggest that FGFR2 is essential for cell survival during early branching morphogenesis.

The tracheal cell death we detected with LysoTracker staining has not been reported using other methods to assay for cell death. To verify that cell death occurs in the trachea at early stages of lung development, we detected dying cells in Dermo1-cre;Fgf10 mutants using an anti-Cleaved Caspase 3 antibody. At E11.5, paraffin sections revealed scattered cell death throughout the length of the trachea (Fig. 4I-N). Additionally, this cell death was seen not only in Dermo1- cre;Fgf10 mutants but also in both Cre-positive and Cre-negative controls. These data confirm the tracheal cell death observed with LysoTracker staining and demonstrate that this tracheal cell death is not due to off-target effects of the Cre allele.

Reduced mesenchymal Fgf10 or epithelial Fgfr2 affects the expression of key lung signaling molecules

Previous findings indicate that epithelial-mesenchymal interactions are critical for lung development and are mediated by complex feedback interactions among signaling pathways including the FGF, SHH and BMP pathways. As most of the evidence supporting feedback regulation is from gain-of-function studies, we sought to test the hypothesis that disruption of the FGF10-FGFR2 pathway in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants alters the expression patterns of other signaling molecules. Because both the SHH and BMP signaling pathways are known to be important during branching morphogenesis (Bellusci et al., 1996; Litingtung et al., 1998; Pepicelli et al., 1998; Li et al., 2008), we further reasoned that potential changes in the activity of these pathways may contribute to the branching defects we observed in the Fgf10 and Fgfr2 mutants in this study.

Shh and patched1 transcripts are reduced in mutant lungs

We first examined Shh transcripts in our mutants because genetic studies indicate that Shh is critical for lung branching. Shh null mice form left and right lung buds that then fail to grow or branch (Litingtung et al., 1998; Pepicelli et al., 1998). In normal controls at E11.25 and E12.5, Shh is expressed throughout the epithelium and is upregulated at the tips of outgrowing branches. Shh expression was similar between Dermo1-cre;Fgf10 mutants and control littermates at E11.25, though the upregulated domains appeared to be somewhat smaller in mutants (Fig. 5A,B). Shh was reduced in mutants at E12.5. Notably, the strong upregulation of Shh normally seen in the distal epithelial tips was reduced in Dermo1-cre;Fgf10 lungs and almost the entire epithelium was expressing Shh at approximately the same level (Fig. 5C,D). In Sftpc-cre;Fgfr2 mutants at these stages, the area in which Shh was upregulated appeared reduced compared to controls. Where outgrowths did form, however, distal upregulation was detected (Fig. 5I-L).

We also analyzed patched 1 (Ptch1) expression in these mutants. Ptch1 expression is upregulated by SHH and therefore serves as a readout of SHH signaling (McMahon, 2000). Normally, Ptch1 is expressed in the mesenchyme surrounding epithelial branches and at high levels around distal branch tips. In Dermo1-cre;Fgf10 mutants, in accordance with diminished Shh expression, Ptch1 appeared to be slightly reduced at E11.25. This reduction was also observed at E12.5 (Fig. 5E-H). In Sftpc-cre;Fgfr2 mutants, consistent with the pattern of Shh expression we observed at E11.25, Ptch1 was expressed at lower levels than in controls (Fig. 5M,N). By E12.5, Ptch1 levels were severely reduced in the mutants (Fig 5O,P). These data provide evidence that disruption of the FGF10-FGFR2 signaling pathway leads to a decrease in SHH signaling.

To complement our loss-of-function data and further test the hypothesis that signaling through the FGF10-FGFR2 pathway regulates Shh expression, we cultured wild type lungs in the presence of recombinant FGF10 protein and assayed for changes in gene expression. After 48 hours of culture, Spry2 expression was upregulated in cells surrounding FGF10-soaked beads (n = 6/7), indicating increased FGF signaling in the tissue associated with the bead (Fig. 5Q,R). Likewise, Shh signal intensity was increased in the tissue surrounding FGF10-soaked beads (n = 6/9) (Fig. 5S,T). No changes in either Spry2 (n = 11/11) or Shh (n = 8/8 ) expression were seen in the presence of BSA-soaked control beads. These results support the conclusion that FGF10 signaling through FGFR2 positively regulates Shh expression in the lung.

Bmp4 expression is decreased in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants

We next assayed for Bmp4 expression in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutant lungs. Previous studies show that overexpression of Bmp4 in the lung epithelium disrupts branching morphogenesis (Bellusci et al., 1996; Hogan et al., 1997; Lebeche et al., 1999) and inactivation of Bmp4 during lung budding leads to hypoplastic lungs (Li et al., 2008). In addition, Bmp4 expression is induced in isolated lung epithelium placed in proximity to an FGF10-coated bead, suggesting that FGF10 signaling promotes Bmp4 expression (Weaver et al., 2000). In normal controls, Bmp4 transcripts are detected at low levels in the mesenchyme and proximal epithelium and at high levels in the distal epithelial tips of outgrowing branches. In Dermo1-cre;Fgf10 mutants at E11.25, Bmp4 expression decreased slightly in the distal tips of the mutant epithelium (Fig. 6A,B), a pattern that persisted at E12.5 (Fig. 6C,D). In Sftpc-cre;Fgfr2 mutants at E11.25, Bmp4 expression decreased in the tips of epithelial buds (Fig. 6I,J). This reduction was more pronounced at E12.5 (Fig. 6K,L), though mRNA was still detected where small epithelial outgrowths formed (Fig. 6M,N). These data indicate that Bmp4 expression is diminished following suppression of FGF signaling.

Figure 6.

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Bmp4 and Fgf10 expression in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants.

(A-T) Gene expression in lungs of indicated genotypes and stages. Arrow in H indicates upregulated Fgf10 expression in the residual rostral lobe mesenchyme of the mutant. M and N are magnified views of bracketed regions in L; arrowheads indicate isolated domains of Bmp4 upregulation. S is a frontal section of the sample shown in R; T is a transverse section of a mutant littermate.

Fgf10 is upregulated and delocalized in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants

Lastly, we examined Fgf10 expression in our mutant lungs to determine whether FGF10 signaling feeds back to regulate its own expression. To do so, we used a probe that hybridizes to regions of the Fgf10 mRNA that flank the deleted exon in the Dermo1-cre;Fgf10 mutant. In control lungs at E10.5, Fgf10 transcript is discretely localized in the mesenchyme at the tips of the outgrowing primary bronchi (Bellusci et al., 1997). At later stages, Fgf10 expression is detected at sites of prospective bud formation. Similar to control lungs, Fgf10 expression in Dermo1-cre;Fgf10 mutants surrounded forming branches at E11.25, E12.5 and E13.5 (Fig. 6E-H and data not shown). However, by E12.5, we noted a substantial upregulation of Fgf10 expression within the rudimentary rostral lobe in those Dermo1-cre;Fgf10 mutant samples that had formed one (Fig. 6H). We observed similar, though more widespread, changes in Fgf10 expression in Sftpc-cre;Fgfr2 lungs. Comparable to controls at E10.5 (data not shown), by E11.25, Fgf10 expression increased at the lobar tips in Sftpc-cre;Fgfr2 mutants. Additionally, the Fgf10 expression domains expanded to include some tissue between those tips (Fig. 6O,P). By E12.5, the domains of Fgf10 expression had expanded farther to include almost the entire peripheral mesenchyme of the mutant lungs (Fig. 6Q-T). Also, expression signal intensity was substantially upregulated. These observations demonstrate that signaling through epithelial FGFR2 indirectly inhibits Fgf10 transcription in the lung mesenchyme.

Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants exhibit changes in expression of P-D lung patterning markers

Several existing mutants that display abnormal branch patterns also exhibit disrupted proximal-distal (P-D) patterning of the epithelium (Weaver et al., 1999; Mucenski et al., 2003). To determine whether P-D patterning is disturbed when FGF10-FGFR2 signaling is reduced, we examined the expression of SRY-box containing gene 9 (Sox9) as a distal marker (Liu and Hogan, 2002; Okubo et al., 2005) and Sox2 as a proximal marker (Ishii et al., 1998; Gontan et al., 2008) of lung patterning.

Sox9 expression is downregulated in mutant lungs

In controls lungs, Sox9 is specifically expressed in distal epithelial cells as well as in the proximal mesenchyme where cartilaginous tracheal rings will form (arrowhead in Fig. 7A). Sox9 expression in Dermo1-cre;Fgf10 mutants was expressed at a slightly lower intensity in the distal epithelium at E11.25 (Fig. 7A,B). Sox9 was reduced overall throughout the epithelium of Dermo1-cre;Fgf10 mutants at E12.5, although its mesenchymal expression was unaltered (Fig. 7C,D). In Sftpc-cre;Fgfr2 mutants at E11.25, Sox9 was comparable to controls (Fig. 7I,J). At E12.5, while mesenchymal expression of Sox9 in the tracheal region remained unchanged in these mutants, distal epithelial expression was clearly reduced (Fig. 7K-M). These results indicate that diminished FGF10-FGFR2 signaling leads to downregulation of Sox9 expression.

Figure 7.

Figure 7

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Expression of P-D lung patterning markers in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants.

(A-R) Expression of P-D markers in lungs of indicated genotypes and stages. Arrowhead in A indicates Sox9 expression in the proximal mesenchyme where cartilaginous tracheal rings will form. M,R are magnified views of bracketed regions indicated in L,Q, respectively. Arrows in G,H and P-R indicate caudal limit of Sox2 expression. Asterisks in G,H and P,Q serve as reference points indicating the position where the main bronchi split. Sox2 is expressed in a longer region of the main bronchi (distance between asterisk and arrow) in the mutant lungs shown in H and Q compared to their respective controls. Arrowheads in M, O and Q indicate isolated domains of expression.

Sox2 expression is expanded in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants

A reduction in Sox9 expression may be the result of having fewer living distal cells due to the role FGF10 signaling plays in promoting survival of the distal epithelium. Alternatively, reduced Sox9 expression may be due to a general shift in P-D patterning if proportionally more epithelial cells adopt a proximal fate while fewer cells adopt a distal fate. To distinguish between these possibilities, we examined the expression of Sox2. In contrast to Sox9, Sox2 is expressed at high levels in the proximal airway epithelium of control lungs but is absent in regions actively engaged in branching morphogenesis, such as in budding distal tips. In Dermo1-cre;Fgf10 mutants at E11.25, Sox2 transcripts were detected at either moderately higher intensities in the epithelium of the primary bronchi (Fig. 7E,F) or were slightly expanded into the distal domains of lobar branches (data not shown). In these mutants at E12.5, the Sox2 expression domain extended somewhat farther from the point where the main bronchi split than it does in controls (asterisks, arrows in Fig. 7G,H). In Sftpc-cre;Fgfr2 mutants, relative to its expression in controls, Sox2 was detected farther along the caudal axis at E11.25 (Fig. 7N,O). This expansion was especially evident at E12.5 (asterisks, arrows in Fig. 7P-R). Additionally, ectopic Sox2 expression was seen in many small, irregular outgrowths that had arisen along the length of the primary bronchi (arrowheads in Fig. 7O,Q). These data suggest that, compared to controls, epithelial cells in more distal positions adopt a proximal fate in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants.

Discussion

In this study, we dissect the roles of the FGF10-FGFR2 signaling pathway during early lung branching morphogenesis by analyzing Fgf10 and Fgfr2 conditional mutants. We show that this signaling pathway affects lung branching by maintaining cell survival and regulating the levels and/or pattern of several signaling molecules that are essential for lung development. In addition, we show that reduction of either Fgf10 in the lung mesenchyme or Fgfr2 in the lung epithelium causes expansion of a proximal lung marker, suggesting that these genes play a role in P-D patterning of the lung.

FGF10 signaling through FGFR2 is essential for normal branching morphogenesis

In both of the mutants characterized in this study, we observed that lobar branching was disrupted, indicating that Fgf10 and Fgfr2 are required for establishing a normal pattern of branching during the early stages of lung development. Though demonstrating a shared requirement for Fgf10 and Fgfr2, these two mutants displayed considerably different phenotypes. Contemplation of the similarities and differences in phenotype between these mutants provides insight into several aspects of the FGF10-FGFR2 signaling dynamic.

In addition to Fgf10, several other FGFs have been implicated in branching morphogenesis, including Fgf9, which is expressed from the initial stages of branching (Colvin et al., 1999; del Moral et al., 2006), and Fgf1 and Fgf7, which are first expressed between E13.5 and E14.5 (Fu et al., 1991; Mason et al., 1994; Finch et al., 1995). Dermo1-cre;Fgf10 mutant lungs often lacked an epithelial branch for the rostral lobe, suggesting that Fgf10 is essential for early branching events subsequent to lung initiation and that other FGFs, such as Fgf9, cannot support epithelial outgrowth in the absence of Fgf10. In contrast to the rostral lobe, branching proceeded in the remaining lobes of Dermo1-cre;Fgf10 mutants. Continued branching could be due to residual Fgf10 expression in these lobes or, alternatively, branching may be promoted by additional FGFs at these later stages. Thus, results from the Dermo1-cre;Fgf10 mutants suggest that Fgf10 is the principle FGF responsible for the initial stages of branching, though it remains possible that additional Fgfs function together with Fgf10 for later rounds of branching.

Residual FGF10-FGFR2 signaling in both mutants provides additional insights into how this pathway impacts lung branching. In Sftpc-cre;Fgfr2 mutants, Fgfr2 transcripts were detected in isolated epithelial cells at E12.5, well after initiation of branching morphogenesis. Unlike Drosophila tracheal branching morphogenesis, where a single FGFR-positive cell can drive outgrowth of a normal branch (Ghabrial and Krasnow, 2006), persistence of isolated FGFR2- positive cells in Sftpc-cre;Fgfr2 mutants did not result in normal branch elongation. This observation suggests that a critical mass of cells capable of responding to FGF10 signaling is required for continuing outgrowth. Similar phenomena, where a minimum number of cells are required to execute a developmental decision, have long been observed in other developmental settings and define the “community effect” (Gurdon, 1988). The Dermo1-cre;Fgf10 mutant phenotype is also consistent with this concept of community effect. A reduced number of branches form in these mutants, possibly because fewer groups of responsive cells are receiving amounts of FGF10 signaling that surpass the threshold required for outgrowth. Interestingly, despite reduced FGF10 signaling, branches that formed in Dermo1-cre;Fgf10 mutants did not appear to be narrower in diameter than they are in controls. This observation suggests that branch diameter is controlled independent of the requirement for Fgf10 in branch outgrowth.

Finally, in both of the mutants analyzed in this study, we detected substantial mesenchymal outgrowth without accompanying epithelial branches. For example, in Dermo1-cre;Fgf10 mutants, mesenchymal outgrowth in the position of the rostral lobe persisted, even though the rostral lobar epithelial branch was often absent. These observations suggest that the outgrowth of an epithelial branch is not essential for mesenchymal outgrowth during lobe initiation.

FGF signaling is required for epithelial cell survival

Both Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants exhibited ectopic cell death, a result consistent with previous findings that suggest FGF signaling is essential for cell survival in other developmental settings (Chi et al., 2003; Verheyden et al., 2005). The amount of cell death seen in Sftpc-cre;Fgfr2 mutants was enhanced and more extensive than that observed in Dermo1- cre;Fgf10 mutants. This phenotypic variation is likely due to differences in the amount of residual FGF signaling between the two mutants. Evidence suggests that the epithelial phenotypes we observed are not due to cell death alone. In both mutants, branching defects were consistently observed as early as E11.25 (Fig. 3). While ectopic cell death also was detected at this stage, it was seen only in approximately half of the mutant lungs. Comparison of these results suggests that the morphogenesis phenotypes precede the increase in cell death.

We propose that increased cell death cannot account for observed changes in gene expression. Several of the genes analyzed, including Shh and Fgfr2 as detected by the TK probe, were expressed in proximal as well as distal lung epithelium. Their expression in the proximal epithelium was not reduced in either mutant compared to control at E12.5 (Fig. 3, 5), even though there was increased cell death in this tissue (Fig. 4). Furthermore, the expression of Fgf10 and Sox2 increased in both mutants at E12.5 (Fig. 6, 7), even though there was increased cell death in their respective expression domains. Thus, we favor the explanation that changes in gene expression are due to disruption of FGF10-FGFR2 regulation of these genes.

FGF signaling regulates important lung development factors

Previous studies led to proposal of a model wherein feedback interactions among Fgf10, Bmp4 and Shh are essential to direct the normal pattern of branch outgrowth (Chuang and McMahon, 2003). Specifically, it was proposed that FGF10 promotes the expression of both Bmp4 and Shh in the distal epithelium. SHH and BMP4 signaling then feeds back to inhibit the expression/function of Fgf10, which leads to division of the Fgf10 expression domain. This lateral shift in Fgf10 expression directs the outgrowth of new epithelial branches. We found that Shh and Bmp4 were downregulated in both Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants. Our in vitro culture results also demonstrated that Shh expression is upregulated in the presence of exogenous FGF10 protein. These data indicate that FGF10-FGFR2 signaling positively regulates epithelial expression of Shh and Bmp4 in the lung, supporting one facet of the model. In support of a second aspect of the model, the proposed feedback regulation of Fgf10, previous data show that BMP4 inhibits FGF10 function (Weaver et al., 2000). Furthermore, transgenic overexpression of Shh in the lung epithelium results in a downregulation of Fgf10 expression while a Shh null mutant exhibits upregulated Fgf10 expression (Bellusci et al., 1997; Pepicelli et al., 1998). Corresponding with decreased expression of Shh and Bmp4 in both Dermo1- cre;Fgf10 and Sftpc-cre;Fgfr2 mutants, we also observed an increase in Fgf10 expression. Thus, our data are consistent with previously published data and with the possibility that SHH and/or BMP4 inhibit Fgf10 expression.

FGF signaling causes the expansion of a proximal lung epithelial marker

Results from both Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants indicate that the FGF10- FGFR2 signaling pathway plays a role in P-D patterning of the lung epithelium. We observed a decrease in expression of the distal marker Sox9 in our mutants. This result is consistent with a previous observation made in a hypomorphic Fgf10 mutant, where expression of distal markers such as Surfactant Protein B and NKX2.1 is reduced at later developmental stages (Ramasamy et al., 2007). Expansion of the proximal marker Sox2 in both mutants analyzed in this study provides more conclusive evidence that FGF10-FGFR2 signaling regulates P-D patterning of the epithelium. One possible explanation for this result is that FGF10 signaling through FGFR2 regulates cell adhesion interactions among, or cell sorting between, distal cells that receive signal versus proximal cells that do not receive signal. However, in light of evidence demonstrating that FGF10 negatively regulates Sox2 expression in the foregut epithelium (Que et al., 2007), we favor a hypothesis wherein the FGF10-FGFR2 pathway regulates P-D patterning by inhibiting proximal fate/gene expression. Whether FGF regulates Sox2 expression directly or indirectly through other signaling pathways remains to be determined. Other FGF-regulated signals, such as the aforementioned BMP4, have been implicated in P-D patterning (Weaver et al., 1999). Furthermore, it has been shown that WNT signaling is decreased in the Fgf10 hypomorphic mutant (Ramasamy et al., 2007). WNT signaling to the lung epithelium has been shown to inhibit proximal lung fate and to promote distal lung fate (Mucenski et al., 2003; Shu et al., 2005; Yin et al., 2008). FGF10-FGFR2 signaling may act through these signaling pathways to establish a normal pattern of epithelial branching.

Experimental Procedures

Generation of Fgf10 and Fgfr2 lung mutants

Generation of mice carrying a conditional allele of Fgf10, in which exon 2 is flanked by loxP sites (Fgf10flox) will be described elsewhere (X. Wang and S. Mansour, in preparation). Fgf10flox/flox mice were mated to mice carrying a Dermo1cre/+ (Twist2tm1(cre)Dor) allele (Yu et al., 2003) and a null allele of Fgf10 (Fgf10tm1Ska) (Sekine et al., 1999) to generate Dermo1cre/+;Fgf10null/flox mutant embryos. Mice carrying a conditional floxed allele of Fgfr2 (Fgfr2tm1Dor) (Yu et al., 2003) were mated to mice carrying a Sftpc-cre transgene (Okubo et al., 2005) to generate Sftpc-cre;Fgfr2flox/flox mutant embryos. Offspring were genotyped using the following PCR primer pairs for Cre: 5′-TGATGAGGTTCGCAAGAACC-3′ and 5′- CCATGAGTGAACGAACCTGG-3′ (product size 420 bp); for Fgf10null: 5′- GCTTCTGCCAATGTATTGCTC-3′ and 5′-TATCGCCTTCTTGACGAGTTCTTCTGA-3′ (product size 750 bp from the null Fgf10 allele); for Fgf10flox: 5′- ATCCTTGGGAGGCAGGATAACC-3′ and 5′-GCAGAGATTGCAAAGGAAGC-3′ (product size 277 bp from the Fgf10flox allele and 174 bp from the wild type Fgf10 gene); for Fgfr2: 5′- TTCCTGTTCGACTATAGGAGCAACAGGCGG-3′ and 5′- GAGAGCAGGGTGCAAGAGGCGACCAGTCAG-3′ (product size 207 bp from the Fgfr2flox allele and 142 bp from the wild type Fgfr2 gene).

Embryo isolation and phenotype analyses

Embryos were dissected from time-mated mice, counting noon on the day the vaginal plug was found as E0.5. As the lungs of Dermo1cre/+;Fgf10flox/+, Dermo1+/+;Fgf10null/flox and Dermo1+/+;Fgf10flox/+ or Sftpc-cre;Fgfr2flox/+, Fgfr2flox/flox and Fgfr2flox/+ littermates were indistinguishable from wild type, they were used as controls for respective Fgf10 or Fgfr2 experiments. To assay for Cre activity through β–galactosidase (β–gal) expression, the ROSA26 reporter line (Soriano, 1999) was introduced into the background of either Dermo1 cre/+ or Sftpc-cre lines. β–gal activity was detected using a standard protocol.

Quantitative RT-PCR

Lungs were collected at E12.5 or E13.5 and separated by lobe. Total RNA was isolated from individual lobes using an RNeasy Kit (Qiagen). cDNA was synthesized using a SuperScript First-Strand cDNA Synthesis System kit (Invitrogen). RT-PCR was carried out using the following primer pairs for β-actin: 5′- CGGCCAGGTCATCACTATTGGCAAC-3′ and 5′- GCCACAGGATTCCATACCCAAGAAG-3′; for Fgf10: 5′-CGGGACCAAGAATGAAGACT-3′ and 5′-AGTTGCTGTTGATGGCTTTG-3.′

RNA in situ hybridization

Whole-mount in situ hybridization using digoxigenin-labeled RNA probes was performed as previously described (Wall and Hogan, 1995). The Fgfr2 exon 8,10 in situ probe was prepared from a plasmid containing the sequences of exons 8 and 10 of the Fgfr2 cDNA. This cDNA was generated via PCR using the primer pair: 5′-AGCTCCAATGCAGAAGTGCTGGC-3′ and 5′- GGATGCGCTTGGTCAGCTTGTGC-3.′ The Fgfr2 TK probe targets regions corresponding to the first kinase domain and the kinase insert (Peters et al., 1992). The Fgf10 probe targets regions corresponding to most of the coding region (Bellusci et al., 1997), including the 3′ end of exon1, all of exon 2 and the 5′ end of exon 3. For all probes, control and mutant lungs at E11.25 or E12.5 were processed together in the same tube. Following hybridization, some samples were embedded in 4% agarose (GenePure LowMelt, ISC BioExpress) in PBT and cut into 50μm sections using a vibratome.

Cell death analysis and Immunofluorescence

To determine the extent of programmed cell death in whole-mount lungs, LysoTracker Red DND-99 (Molecular Probes) staining was employed to label apoptotic cells using a modified protocol (Zucker et al., 1999). To determine the extent of cell death in the trachea, immunofluorescence staining was performed on paraffin sections. Tissues were fixed in 4% PFA, dehydrated into methanol, infiltrated with paraffin and cut into 7 μm sections. Sections were hydrated and boiled in 10mM sodium citrate, pH 6.0, for 20 min, then washed with PBS. Tissues were blocked for 1 hr at room temperature with 5% normal goat serum (NGS)/PBS plus 0.2% Triton X-100 (PBTr), then incubated overnight at 4°C with primary antibody in diluted block solution (1% NGS/PBTr). A rabbit anti-Cleaved Caspase 3 antibody (Cell Signaling Technology, 1:500 dilution) was used to label dying cells and a rabbit anti-E-Cadherin antibody (Cell Signaling Technology, 1:100 dilution) was used to label the tracheal epithelium on adjacent serial sections. Tissues were washed with PBS and incubated for 30 min at room temperature with Cy3-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, 1:250 dilution) in 1% NGS/PBTr. Sections were washed with PBS, stained with DAPI to label cell nuclei and washed again. Sections were mounted with Vectashield mounting medium (Vector Labs) and visualized by immunofluorescence.

In vitro lung culture

Lungs from Swiss Webster mice were harvested at E11.5 and placed on a Nucleopore Track-Etch membrane (Whatman, 8μm). Heparin beads were soaked in recombinant human FGF10 protein (PeproTech, 100 ng/μL) or 1% BSA and implanted in the right lung. Membranes were cultured at the air/liquid interface on 0.5mL GIBCO BGJb media (Invitrogen) supplemented with ascorbic acid (Sigma) and GIBCO Antibiotic-Antimycotic (Invitrogen) in a 4-well Nunclon dish (Nunc, Thermo Fisher Scientific). Cultures were incubated at 37°C in 5% CO2 for 48 hours, fixed in 4% PFA and processed for in situ hybridization.

Acknowledgments

We thank Dr. David Ornitz for sharing Dermo1cre and Fgfr2flox mice, Dr. Brigid Hogan for sharing Sftpc-cre mice and Drs. Hideyo Ohuchi, Nobuyuki Itoh and Saverio Bellusci for supplying Fgf10 null mice. We are grateful to X. Wang (Mansour lab) for generating the Fgf10flox mice. We thank Drs. S. Bellusci, D. Epstein, P. Gray, V. Lefebvre, G. Minowada, K. Peters and J. Wozney for plasmids used in analysis of phenotypes. We appreciate the many insightful discussions we had with members of the Sun lab. We are indebted to Amber Lashua and Minghui Zhao for animal husbandry and technical assistance and to Jen Heinritz for genotyping help. This work was supported by a Burroughs-Wellcome career award #1002361 and a Young Investigator Award from the University of Wisconsin Medical Education Research Committee (both to X.S.).

Grant Information: Burroughs-Wellcome career award #1002361; Young Investigator Award from University of Wisconsin Medical Education Research Committee.

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