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Dev Biol. Author manuscript; available in PMC 2009 August 1.
Published in final edited form as:
Dev Biol. 2008 August 1; 320(1): 149–160.
Published online 2008 May 10. doi: 10.1016/j.ydbio.2008.04.038.
PMCID: PMC2586150
NIHMSID: NIHMS64590
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Misexpression of ELF5 Disrupts Lung Branching and Inhibits Epithelial Differentiation
David E. Metzger,*‡ Mildred T. Stahlman,† and John M. Shannon*‡1
*Division of Pulmonary Biology, Cincinnati Children’s Hosptial Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039
‡Molecular and Developmental Biology Graduate Program, University of Cincinnati College of Medicine, Cincinnati, OH
†Department of Pediatrics, Vanderbilt University Medical Center
1Corresponding author: (513) 636-2938; (513) 636-7868 FAX); e-mail: john.shannon/at/cchmc.org
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Abstract
ELF5, an Ets family transcription factor found exclusively in epithelial cells, is expressed in the distal lung epithelium during embryogenesis, then becomes restricted to proximal airways at the end of gestation and postnatally. To test the hypothesis that ELF5 represses distal epithelial differentiation, we generated a transgenic mouse model in which a doxycycline inducible HA-tagged mouse Elf5 transgene was placed under the control of the lung epithelium-specific human SFTPC promoter. We found that expressing high levels of ELF5 during early lung development disrupted branching morphogenesis and produced a dilated epithelium. The effects of ELF5 on morphogenesis were stage dependent, since inducing the transgene on E16.5 had no effect on branching. ELF5 reduced expression of the distal lung epithelial differentiation markers Erm, Napsa and Sftpc, and type II cell ultrastructural differentiation was immature. ELF5 overexpression did not induce the proximal airway epithelial markers Ccsp and Foxj1, but did induce expression of p63, a marker of basal cells in the trachea and esophagus. High ELF5 levels also induced the expression of genes found in other endodermal epithelia but not normally associated with the lung. These results suggest that precise levels of ELF5 regulate the specification and differentiation of epithelial cells in the lung.
Keywords: lung development, Ets, ELF5, gene transcription, lung differentiation
The mouse lung first appears at E9.5 as two outgrowths from the foregut endoderm that invade the adjacent splanchnic mesoderm. Following this initial event, the foregut endoderm undergoes stereotypic branching within the lung mesenchyme to form the basic structure of the pulmonary tree. Later in gestation and in early postnatal life, the lung goes through stages of distal airway expansion, distal and proximal airway cellular differentiation, septation and alveolarization. These events are all dependent on the correct spatiotemporal expression and regulation of genes and proteins such as transcription factors and secreted factors (Cardoso and Lu, 2006; Maeda et al., 2007; Warburton et al., 2005).
Secreted factors are known to be responsible for intercellular interactions that occur between the lung epithelium and mesenchyme (Shannon and Hyatt, 2004). For example, FGF10 secreted by the lung mesenchyme signals through FGFR2b in the epithelium and acts as a chemoattractant on the epithelium to drive branching morphogenesis (Bellusci et al., 1997; Hyatt et al., 2004; Min et al., 1998; Ohuchi et al., 2000; Park et al., 1998; Sekine et al., 1999) and to maintain a population of progenitors that later differentiate into the peripheral cell types of the lung (Hokuto et al., 2003; Perl et al., 2002b; Ramasamy et al., 2007). Other factors also influence the branching epithelium, notably BMPs and Wnts, which are important for distal/proximal specification of the epithelium as well as maintenance of lung epithelial progenitors (Bellusci et al., 1996; Dean et al., 2005; Eblaghie et al., 2006; Hyatt et al., 2002; Hyatt et al., 2004; Mucenski et al., 2003; Okubo and Hogan, 2004; Shu et al., 2005; Weaver et al., 2000; Weaver et al., 1999).
Secreted ligands such as FGF10 bind their receptors and initiate signaling cascades that activate downstream target genes. In a previous study we identified Elf5 (Ese2) as a gene activated in the embryonic lung epithelium by FGF signaling (Metzger et al., 2007). ELF5 is a member of the Ese (Epithelial Specific Ets) family that was originally reported as being expressed in differentiating keratinocytes and branching epithelia such as salivary gland, prostate, mammary gland and kidney (Oettgen et al., 1999; Zhou et al., 1998). We found that Elf5 expression was dynamic throughout lung development. During early branching morphogenesis Elf5 expression was restricted to the distal lung epithelium, then became enriched in the proximal airway epithelium and diminished distally at the end of gestation and into postnatal lung development. From these results we hypothesized that ELF5 may initially act to regulate epithelial branching, then later in development increased ELF5 in the proximal epithelium may negatively regulate distal epithelial gene expression and differentiation. To test these hypotheses, we have now performed gain-of-function studies by conditionally expressing HA-tagged ELF5 under the control of the lung epithelium-specific human SFTPC promoter. We found that ELF5 misexpression disrupts branching morphogenesis and negatively regulates distal lung epithelial differentiation. Microarray analysis of lungs overexpressing ELF5 reveals the repression of some type II cell-specific genes, but the induction of others. High levels of ELF5 expression also induced the expression of genes not typically found in the distal lung epithelium, suggesting that ELF5 can influence eventual cell fate.
Generation of HA-Elf5 mice
The full-length mouse Elf5 cDNA was amplified from E12.5 lung by RT-PCR using primers 5’-CGGGATCCCGATGGCGTACCCATACGACGTGCCTGACTACGCCTCCCTCATGTTGGAACTCCGTA-3’ and 5’-CCATCGATGGCCATCAAATGAGCCTGGTGT-3’ to add an N-terminal HA-tag to the ELF5 protein and 5’ BamHI and 3’ ClaI cut sites to facilitate cloning. The PCR product was then ligated into the rtTA inducible pTRE-Tight vector (Clontech, Mountain View, CA) digested with BamHI and ClaI. The pTRE-Tight-HA-Elf5 vector was cut with XhoI to give a fragment that was used for injection into FVB/N oocytes by the Cincinnati Children’s Hospital Research Transgenic Core Facility. Genotyping was performed by PCR on genomic DNA using 2 different primer sets. The first primer set, 5’-CGTATGTCGAGGTAGGCGTGTA-3’ and 5’-TCAGGTTCAGGGGGAGGTGTG-3’, was specific for the pTRE-Tight promoter while the second primer set, 5’-TACCCATACGACGTGCCTGAC-3’ and 5’-CCATCGATGGCCATCAAATGAGCCTGGTGT-3’ was specific for the HA-Elf5 transgene.
Conditional expression of the HA-Elf5 transgene to lung epithelium was accomplished by breeding SFTPC-rtTA mice (Perl et al., 2002a) to HA-Elf5 mice (hereafter SFTPC/Elf5 mice) and then treating the mice with doxycycline (Dox). Except as noted below, pregnant dams were treated with Dox from the day of conception. Transgene expression was traced using anti-HA immunohistochemistry (IHC). Littermates bearing a single transgene (SFTPC-rtTA or HA-Elf5) were used as controls.
Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde or formalin, embedded in paraffin, and 5 µm sections were cut for IHC. Primary antibodies used were: HA (rabbit, 1:200, Santa Cruz Biotech; Santa Cruz, CA), mouse IgG1 (1:200, Cell Signaling; Danver, MA), ELF5 (goat, 1:50, Santa Cruz Biotech), phosphohistone H3 (pHH3; mouse, 1:500, Santa Cruz Biotech), Cleaved Caspase3 (rabbit, 1:5000; R & D; Minneapolis, MN), p63 (mouse 4A4, 1:50, Santa Cruz Biotech), Pro-SP-C (rabbit, 1:1000 and guinea pig, 1:1000), TTF1 (rabbit, 1:500), FoxA1 (guinea pig, 1:1000), FoxA2 (rabbit, 1:1000), CCSP (rabbit, 1:1000), FoxJ1 (rabbit, 1:5000). The antibodies against Pro-SP-C, TTF1, FoxA1, FoxA2, CCSP and FoxJ1 were generous gifts from Dr. Jeffrey Whitsett. Double IHC staining was performed using the ELF5 antibody with the rabbit Pro-SP-C antibody, or using the HA antibody with the guinea pig Pro-SP-C antibody. Biotinylated secondary antibodies were used with the ABC Vectastain kit (Vector Laboratories; Burlingame, CA). DAB with nickel enhancement and the AEC peroxidase substrate kit (Vector Laboratories) were used for visualization.
Proliferation analysis
The percentages of pHH3 positive epithelial and mesenchymal cells in E16.5 SFTPC-rtTA and SFTPC/Elf5 lungs were counted after pHH3 staining. Specifically, three 20X micrographs of representative fields were taken from three different control or double transgenic lungs, and pHH3 positive and negative cells were counted. A minimum of 1000 epithelial cells and mesenchymal cells were counted per slide. Proliferation data were statistically analyzed using a Student’s t-test on GraphPad Prizm version 4.0 (GraphPad Software, San Diego, CA).
Microarray analysis
Cysts from the lungs of E16.5 SFTPC/Elf5 fetuses and spatially similar areas from SFTPC-rtTA control lungs were dissected, and RNA was isolated using an RNeasy Micro Kit (Qiagen, Valencia, CA). RNA from three SFTPC/Elf5 lungs and three SFTPC-rtTA littermate lungs were used in the analysis. The microarray analysis was performed as previously described (Metzger et al., 2007). Differentially expressed genes between experimental and control samples were determined by performing a Student’s t-test (p ≤ 0.05) and filtering for genes that increased or decreased at least 2-fold. Differentially expressed genes were further compared to genes from a previously published microarray (Matsuzaki et al., 2006) using genes with two present calls in wild-type adult type II cells.
RT-PCR and Quantitative PCR
Adult alveolar type II cells were isolated by dispase digestion and differential adherence (Rice et al., 2002) and MLE15 cells, which are immortalized distal epithelial cells, were grown in HITES medium (Wikenheiser et al., 1993). RNA for RT-PCR was isolated and reverse transcribed into cDNA by standard methods. The primers used for RT-PCR were as follows:
  • Elf5, forward: 5’-GGACTCCGTAACCCATAGCA-3’
  • reverse: 5’-TACTGGTCGCAGCAGAATTG-3’;
  • Sftpc, forward: 5’-CATACTGAGATGGTCCTTGAG-3’
  • reverse: 5’-TCTGGAGCCATCTTCATGATG-3’
  • β-actin, forward: 5’-TGGAATCCTGTGGCATCCATGAAAC-3’
  • reverse: 5’-TAAAACGCAGCTCAGTAACAGTCCG-3’
For quantitative PCR (qPCR), we dissected cysts from E16.5 SFTPC/Elf5 lungs treated with doxycycline from E0 – E16.5, isolated the RNA, and generated cDNA. Spatially similar areas from nontransgenic or single transgenic SFTPC-rtTA or HA-Elf5 littermate lungs served as controls. Analysis was done on 3–5 independent tissue isolations. We performed qPCR using Taqman probe and primer sets (Applied BioSystems, Foster City, CA) specific for Elf5 (Assay ID: Mm00479832_m1), Ese3/Ehf (Mm00468193_m1), Spdef (Mm00600221_m1), Ese1/Elf3 (Mm00468224_m1), Sftpc (Mm00488144_m1), Erm/Etv5 (Mm00465816_m1), Pea3/Etv4 (Mm01245872_m1), Spink5 (Mm00511522_m1), Spink4 (Mm00803437_m1), Scgb3a1 (Mm00446493_m1), Scgb3a2 (Mm00504412_m1), TFF3 (Mm00495590_m1) and p63/Trp63 (Mm00495788_m1), and Napsa (Mm00492829_m1). A probe and primer set for 18s rRNA was used as the normalization standard. The PCR reactions and relative quantifications were performed using 25ng of cDNA per reaction in a 7300 Real-Time PCR Sytem (Applied BioSystems). Relative quantification data from qPCR analysis were statistically analyzed using Student’s t-tests on GraphPad Prizm version 4.0.
Expression of ELF5 during lung development
We previously reported that Elf5 mRNA is initially expressed in the distal tip lung epithelium at E11.5, becomes highly expressed in the proximal airway epithelium as development progresses, then decreases distally (Metzger et al., 2007). We have now extended these observations to determine expression of ELF5 protein in lungs by IHC and found an identical pattern throughout gestation. ELF5 was localized to the nuclei of epithelial cells in the branching distal tips, but absent from the proximal epithelium at E14.5 (Fig. 1AFig. 1). At E15.5 ELF5 was expressed in the proximal epithelium as well as the distal epithelium (Fig. 1BFig. 1), and there was a low level of the distal epithelial marker Pro-SP-C expressed in the distal tips (Fig. 1CFig. 1). E16.5 lungs showed increased ELF5 expression in the proximal airway epithelium and continued ELF5 expression in the distal epithelium (Fig. 1DFig. 1), while Pro-SP-C, a marker specific to distal lung epithelium in the fetus and to alveolar type II cells postnatally, was still expressed at a relatively low level (Fig. 1EFig. 1). At E17.5, cells of the airway epithelium showed strong staining for ELF5 compared to cells in the lung parenchyma (Fig. 1G,IFig. 1), and Pro-SP-C was strongly expressed in presumptive alveolar type II cells (Fig. 1HFig. 1). To determine the identity of the ELF5 positive cells in the lung parenchyma at E17.5, we performed double IHC for ELF5 and Pro-SP-C and found that ELF5 positive cells in the lung parenchyma were presumptive type II cells (Fig. 1KFig. 1). Because alveolarization continues after birth, we further evaluated the co-expression of ELF5 and Pro-SP-C protein in the lungs of postnatal day 6 (PND6) and 4-week old mice (Fig 1L,MFig. 1). ELF5 was undetectable in Pro-SP-C positive type II cells at these time points, which is consistent with our previous ISH results (Metzger et al., 2007), and a previous study that used a different ELF5 antibody (Lapinskas et al., 2004). We observed some apical staining with the ELF5 antibody, but determined that this was non-specific (Fig. 1JFig. 1). Because the IHC data showed that distal expression of Elf5 decreased as Sftpc increased, we performed RT-PCR for Elf5 and Sftpc on isolated adult type II cells and on the immortalized distal epithelial cell line MLE15. The results (Fig. 1FFig. 1) revealed an inverse relationship between Elf5 and Sftpc expression. Although the RT-PCR data indicate that Elf5 is present at low levels in adult type II cell preparations, this may be due to the fact that isolated type II cell preparations typically contain some contaminating proximal epithelial cells. Overall the data indicate that Elf5 expression is very low in fully differentiated distal epithelial cells.
Fig. 1
Fig. 1
Fig. 1
Endogenous expression of ELF5 and Pro-SP-C protein expression during lung development. ELF5 is expressed at the tips of branching lung epithelium (arrows) at E14.5 (A). ELF5 expression continues in branching epithelium (arrows) at E15.5 but is also expressed (more ...)
Misexpression of ELF5 in the distal epithelium disrupts lung branching morphogenesis
Our previous study (Metzger et al., 2007) indicated that the distal epithelial expression of Elf5 during the pseudoglandular stage of lung development was regulated by FGF signaling and was specifically downstream of FGFR2b. We hypothesized that ELF5 might play a downstream role in FGF signaling that is important for lung morphogenesis and differentiation. In order to investigate the function of ELF5 during branching morphogenesis and its role in distal epithelial differentiation, we used the 3.7 kb human SFTPC promoter to direct expression of a Dox-inducible HA-Elf5 transgene to the distal lung epithelium (SFTPC/Elf5 mice). Recently, a longer isoform of ELF5 has been described that contains an additional 31 amino acids at the N-terminus (Choi and Sinha, 2006); these isoforms were designated as ESE2 and ESE2L (with the additional 31 amino acids). Our misexpressed ELF5 protein corresponds to the ESE2 isoform.
We identified a transgenic line in which Elf5 was expressed at a high level; at E16.5 the lungs of these mice showed a 16-fold increase over endogenous Elf5 expression (Fig. 8Fig. 8). When we examined the effects of increased ELF5 expression on E14.5 we found that, compared to the typical fine branches in lungs of single transgenic littermate control animals (Fig. 2AFig. 2), the lungs from double transgenic embryos that were treated with Dox from conception had dilated distal branches (Fig. 2BFig. 2). This phenotype was not uniform throughout the lungs of double transgenic embryos, because branching appeared normal in some areas. When we traced expression of the HA-ELF5 fusion protein using HA IHC, we found that the HA-Elf5 transgene was expressed in the distal lung epithelium (Fig. 2IFig. 2), and that its expression appeared to be mosaic, which likely contributed to the variability in phenotype. Lungs from E16.5 embryos treated with Dox from conception gave similar phenotypes (Fig. 2C–FFig. 2), with the distal epithelium in SFTPC/Elf5 lungs displaying dilated, cystic branches (Fig. 2D,F,HFig. 2) compared to the fine branches of single transgenic controls (Fig. 2C,E,GFig. 2). We confirmed expression of the HA-ELF5 fusion protein in the E16.5 SFTPC/Elf5 lung epithelium by IHC (Fig. 2JFig. 2) and again observed mosaic expression.
Fig. 8
Fig. 8
Fig. 8
E16.5 SFTPC/Elf5 lungs were compared to controls by qPCR for expression of other Ets transcription factors (n = 5) Sftpc (n = 5) and Napsa (n = 3). Elf5 was significantly (p < 0.005) increased in SFTPC/Elf5 lungs, as were the Epithelial Specific (more ...)
Fig. 2
Fig. 2
Fig. 2
Misexpression of ELF5 in distal lung epithelial cells disrupts branching morphogenesis at E14.5. Control lungs from an E14.5 SFTPC-rtTA embryo show typical fine branches (A), whereas the lungs from an E14.5 SFTPC/Elf5 embryo display areas of epithelial (more ...)
High ELF5 expression inhibits epithelial proliferation
Proper branching morphogenesis requires a high rate of cellular proliferation to generate the number of cells required for tissue expansion, and a cystic phenotype may result in the absence of sufficient proliferation. One possible reason for the inhibition of branching we observed in SFTPC/Elf5 lungs is that proliferation was decreased. We therefore assessed the rate of proliferation in lungs from E16.5 SFTPC/Elf5 and control mice by IHC for pHH3, a marker for dividing cells (Fig. 3A,BFig. 3). Epithelial cells in the periphery of single transgenic control lungs stained strongly for pHH3 (Fig. 3AFig. 3), indicating a high rate of proliferation. We observed much lower epithelial pHH3 staining in the lungs of SFTPC/Elf5 fetuses (Fig. 3BFig. 3). Quantification of the percentage of proliferating epithelial cells revealed a significant decrease of epithelial proliferation in double transgenic lungs (Fig. 3CFig. 3). There was no significant change in mesenchymal proliferation between control and experimental lungs (Fig. 3DFig. 3), demonstrating that the effect of high ELF5 expression on proliferation was specific to the epithelium. To determine if the HA-ELF5 fusion protein was inducing cell death in double transgenic lungs, we stained for the apoptosis marker cleaved caspase 3. We saw very few apoptotic cells in either control (Fig. 3EFig. 3) or double transgenic lungs, even in areas of high transgene expression (Fig. 3F’Fig. 3).
Fig. 3
Fig. 3
Fig. 3
ELF5 overexpression inhibits epithelial proliferation. IHC for the proliferation marker phospho-Histone H3 (pHH3) in E16.5 SFTPC-rtTA (A) and SFTPC/Elf5 (B) lungs shows decreased epithelial staining in SFTPC/Elf5 lungs. Quantification of the percentage (more ...)
High ELF5 expression inhibits distal epithelial differentiation
The results in Fig. 1Fig. 1 demonstrate that although distal epithelial expression of ELF5 is high during branching morphogenesis, it is extinguished postnatally. In contrast, proximal epithelial ELF5 expression is low early in gestation, but progressively increases in late gestation and after birth. One possible explanation for this dynamic expression pattern is that ELF5 plays a role in suppressing distal differentiation. To determine the effects of ELF5 overexpression on distal epithelium differentiation, we evaluated the lungs of E16.5 embryos that had been exposed to Dox since conception for markers of lung epithelial differentiation (Fig. 4Fig. 4). As expected, we found that Pro-SP-C protein was uniformly expressed in distal epithelial cells in control lungs (Fig. 4AFig. 4). We did not detect Pro-SP-C expression in the dilated cysts of SFTPC/Elf5 lungs, although histologically normal lung tissue in the same section expressed the protein (Fig. 4BFig. 4). We also assessed the expression of other proteins known to be important in distal lung epithelial differentiation. We found that virtually all of the epithelial cells in SFTPC/Elf5 lungs were positive for FOXA1 (Fig. 4E,FFig. 4) and FOXA2 (Fig. 4G,HFig. 4), which are markers of endoderm. Similarly, all of the epithelial cells stained positive for TTF1 (Fig. 4C,DFig. 4), indicating that the cells had entered the respiratory lineage.
Fig. 4
Fig. 4
Fig. 4
Effects of ELF5 misexpression on gene expression in E16.5 lungs. Sections of SFTPC-rtTA control lungs (A) and SFTPC/Elf5 lungs (B) stained for Pro-SP-C protein show that it is not detectable in the cystic epithelium of SFTPC/Elf5 lungs (asterisks), although (more ...)
We next examined lungs at E18.5, when saccule development has begun in the normal lung (Fig. 5AFig. 5). We found the same morphology of dilated epithelial cysts in SFTPC/Elf5 lungs (Fig. 5BFig. 5). The dilated cysts persisted in the lungs of adults (Fig. 5DFig. 5), whereas controls were extensively alveolarized (Fig. 5CFig. 5). Similar to lungs from E14.5 and E16.5 embryos, the extent of phenotype was variable, with some lobes in adult lungs apparently unaffected (data not shown). Because Pro-SP-C was undetectable in the cysts of E18.5 SFTPC/Elf5 lungs, we examined these regions by electron microscopy for evidence of changes in type II cell ultrastructural differentiation. We found that SFTPC-rtTA control lungs contained many glycogen-laden type II cells with microvilli and cytoplasmic lamellar bodies, which are the storage organelle for pulmonary surfactant. Secreted surfactant was present in the alveolar lumina (Fig. 5EFig. 5). In contrast, the epithelial cells forming the dilated cysts of SFTPC/Elf5 E18.5 lungs were undifferentiated. Low cuboidal epithelial cells contained copious glycogen deposits, but did not contain lamellar bodies. Secreted surfactant was never seen in luminal spaces of cystic regions. The interstitial space adjacent to the epithelial lining was very loosely organized, and the resident fibroblasts contained many lipid droplets (Fig. 5FFig. 5).
Fig. 5
Fig. 5
Fig. 5
Histology of E18.5 SFTPC-rtTA (A) and SFTPC/Elf5 (B) lungs shows that expression of ELF5 transgene disrupts acinar formation, and this persists in adult control (C) and double transgenic (D). Electron micrographs of E18.5 SFTPC-rtTA control (E) and SFTPC/Elf5 (more ...)
Pro-SP-C immunostaining on lungs from fetuses continuously exposed to Dox through day E18.5 (DOX E0–E18.5), gave results similar to those observed on E16.5: Pro-SP-C expression was undetectable in the dilated cysts of SFTPC/Elf5 lungs, whereas control lungs and the histologically normal parenchyma of double transgenic lungs strongly expressed the protein (Fig. 6A,BFig. 6). Accordingly, HA IHC of these lungs revealed that the HA-Elf5 transgene expression was only seen in the epithelium of dilated cysts and was absent from unaffected areas (Fig. 6KFig. 6).
Fig. 6
Fig. 6
Fig. 6
ELF5 represses SP-C expression in E18.5 lungs. IHC for Pro-SP-C in the lungs of SFTPC/Elf5 embryos that had been exposed to Dox since conception (DOX E0–E18.5) reveals a lack of Pro-SP-C in the epithelial cysts (*) (B), whereas SFTPC-rtTA control (more ...)
In order to determine if the effects of Elf5 overexpression were dependent on the developmental stage at which the transgene was induced, we waited until E16.5 to treat dams with Dox (DOX E16.5–E18.5). This delayed induction of the HA-Elf5 transgene until the end of the pseudoglandular stage, when patterning of the pulmonary tree has been completed, but formation of acini and saccules is still ongoing. We found that the morphology of DOX E16.5–E18.5 embryos was very similar lung morphology to control littermates. We did not observe cystic dilation of the distal epithelium, even when transgene expression was widespread (Fig. 6LFig. 6). However, we still observed decreased Pro-SP-C expression in SFTPC/Elf5 lungs (Fig. 6HFig. 6) compared to those of single transgene littermate controls (Fig. 6GFig. 6). Double IHC staining revealed that HA positive cells did not co-express Pro-SP-C (Fig. 6I,JFig. 6), confirming that high ELF5 levels in late gestation still repressed Pro-SP-C expression.
Since ELF5 is highly expressed in the airway epithelium in late gestation we speculated that ELF5 might induce genes specific to the proximal airway epithelial cell differentiation. We therefore evaluated CCSP, a marker of Clara cells, in DOX E0–E18.5 lungs by IHC. We found that endogenous CCSP was present in the proximal airway epithelium of both DOX E0–E18.5 and control lungs (Fig. 6C,DFig. 6). Notably, the distal cystic epithelium of SFTPC/Elf5 lungs was negative for CCSP (Fig. 6DFig. 6). In parallel with these results, expression of the ciliated cell marker, FOXJ1, was restricted to the proximal airway epithelium in both control and experimental lungs, and was not seen in the cystic epithelium (Fig. 6E,FFig. 6).
Microarray analysis
The lack of Pro-SP-C expression and the appearance of immature cuboidal epithelial cells in double transgenic lungs suggested that distal epithelial cell differentiation was impaired; however, the fate of the cells in the cysts was unknown. To gain some insight into the identity of the cells, we isolated cystic regions of E16.5 SFTPC/Elf5 lungs and spatially similar regions from the lungs of SFTPC-rtTA littermates for microarray analysis. Since transcriptional targets of ELF5 and other ESE transcription factors have not been well characterized (Choi and Sinha, 2006), we hypothesized that microarray analysis would provide candidate ELF5 target genes. We found that overexpression of ELF5 activated 202 genes at least 2-fold and decreased the expression of 123 genes. These included genes involved in transcriptional regulation, transport, receptor activity, intracellular signaling, adhesion, and a number of secreted proteins (Supplement).
Since differentiation of type II cells in E18.5 SFTPC/Elf5 lungs was defective, we compared the list of differentially expressed genes to those present in isolated wild-type adult mouse type II cells; 40 of the 123 genes that decreased with ELF5 overexpression were present in differentiated type II cells (Table 1). Some of these genes are associated with type II cell differentiation. Both Erm (Etv5) and Napsa, which are expressed in differentiated type II cells, were decreased in the microarray, a result we confirmed by qPCR (Fig 8Fig. 8). Conversely, 92 of 202 genes that increased with ELF5 overexpression were present in isolated type II cells (Table 2). Some of these genes, such as Sftpd, Spink5, and Scgb3a1, are not only present in type II cells but are also expressed in the proximal airway epithelium. The increases in Spink5 and Scgb3a1 were confirmed by qPCR (Fig. 7AFig. 7).
Table 1
Table 1
Genes present in adult type II cells but decreased with Elf5 overexpression.
Table 2
Table 2
Genes present in adult type II cells and upregulated by Elf5 everpression.
Fig. 7
Fig. 7
Fig. 7
Cystic regions of E16.5 SFTPC/Elf5 lungs were compared by microarray to spatially similar areas of SFTPC-rtTA control lungs to identify upregulated genes. qPCR for expression of 6 of the genes identified-- Spink5, Spink4, Scg3a1, Scgb3a2, Tff3 and p63 (more ...)
ELF5 also induced the expression of genes not typically associated with the distal lung, such as Spink4, Guca2a, Tff3 and Dcpp (Supplement); we verified the induction of Spink4 and Tff3 by qPCR (Fig. 7AFig. 7). We identified other genes increased in SFTPC/Elf5 lungs that are not present in type II cells but are expressed in the proximal airway epithelium (Supplement), such as Scgb3a2 (Reynolds et al., 2002), whose upregulation in SFTPC/Elf5 lungs was verified by qPCR (Fig. 7AFig. 7). ELF5 overexpression also induced the expression of the basal cell marker, p63, which is normally found in the pseudostatified epithelium of the trachea and bronchus (Fig. 7HFig. 7) as well as the stratified esophageal epithelium. Because we observed Elf5 expression in these tissues at E17.5 (Fig. 7F,GFig. 7), we analyzed p63 expression in DOX E0–E16.5 SFTPC/Elf5 lungs by IHC. We found that p63 was detectable in the cysts of double transgenic lungs (Fig. 7CFig. 7), while control lungs lacked p63 expression in the distal epithelium (Fig. 7BFig. 7). Similarly, p63 was also induced in DOX E16.5–E18.5 double transgenic E18.5 lungs (Fig. 7EFig. 7), but not in controls (Fig. 7DFig. 7). Esophageal epithelium, previously reported to have a high expression of p63 (Daniely et al., 2004; Que et al., 2007), was used as an internal positive control for experimental samples (Fig. 7B,D, insetsFig. 7).
ELF5 regulates the expression of other Ets factors in the lung
Along with the predicted increase of Elf5, another Epithelial-Specific Ets factor, Ese3, was increased in the microarray and was also present in type II cells (Table 2); these data were confirmed with qPCR (Fig.8Fig. 8). We then evaluated by qPCR the expression of other Epithelial Specific Ets factors, Spdef and Ese1, which were not changed as determined by microarray. We found that Spdef, previously described as a marker for goblet cells in the airway epithelium (Park et al., 2007), was significantly increased in SFTPC/Elf5 lungs, but Ese1 expression did not change (Fig. 8Fig. 8). Erm, which has previously been shown to be highly expressed in type II cells, to directly activate the Sftpc promoter, and to be critical for distal epithelium differentiation (Lin et al., 2006; Liu et al., 2003) was decreased in both microarray and qPCR analyses. We also found that Sftpc expression was reduced by microarray and qPCR (Fig. 8Fig. 8), supporting our IHC results (Fig. 6Fig. 6). Pea3, which is thought to play a redundant role to Erm (Liu et al., 2003), did not change (Fig. 8Fig. 8).
ELF5 expression during lung development
We previously reported that Elf5 is expressed in the distal tip lung epithelium at E11.5, becomes more highly expressed in the proximal airway epithelium as development progresses, then decreases distally at the end of gestation and postnatally (Metzger et al., 2007). We have now extended these observations to show that ELF5 protein expression follows a parallel pattern throughout gestation. As late as E17.5, the distal epithelial cells that expressed ELF5 co-expressed Pro-SP-C, identifying them as presumptive type II cells. ELF5 expression was no longer detectable in Pro-SP-C positive type II cells after birth, which is consistent with our ISH results (Metzger et al., 2007), and a previous study using a different ELF5 antibody (Lapinskas et al., 2004). These data indicate that Elf5 expression is very low in differentiated type II cells, and suggested the possibility that ELF5 may play a role in negatively regulating Sftpc. Although our microarray data (Table 2) indicate that Elf5 is present in type II cell preparations, our RT-PCR results indicate that the expression is at low levels. We believe that this low level of expression is due to the fact that isolated type II cell preparations typically contain some contaminating proximal epithelial cells, which are Elf5 positive in the postnatal lung.
ELF5 overexpression causes lung branching defects
Our previous study indicated that the distal epithelial expression of Elf5 during the pseudoglandular stage of lung development was regulated by FGF signaling, and was specifically downstream of FGFR2b. Because of the importance of FGF10, which uses FGFR2b as its primary receptor, in lung development (Min et al., 1998; Sekine et al., 1999), we hypothesized that ELF5 might act downstream in regulating morphogenesis and differentiation. We tested this hypothesis by overexpressing ELF5 using the human SFTPC promoter, which is active in the epithelium from the beginning of mouse lung development (Perl et al., 2002b). We found that misexpressed ELF5 disrupted branching morphogenesis, as evidenced by the formation of dilated cysts in the distal epithelium. This phenotype, which we detected as early as E14.5, persisted throughout gestation and into postnatal life. HA IHC verified that the transgene was being expressed in the distal epithelium, but its expression was mosaic, causing a partially cystic phenotype. We also observed that only a subset of cells in the cystic regions were transgene positive as gauged by HA IHC. The basis for this mosaic expression pattern is unknown. One possible explanation stems from our observation that ELF5 decreases Sftpc expression (Fig. 4Fig. 4): since we drove ELF5 expression using the SFTPC promoter, it could have the secondary effect of reducing its own expression, but only in a subset of cells. Alternatively, transgene expression may have only been high enough to detect in a subset of cells, and these affected their neighbors through crosstalk via secreted factors. The effects of ELF5 overexpression on lung branching were restricted to a window during development, however, because morphogenesis was unaffected when the transgene was induced beginning on E16.5, when basic patterning of the lung has been mostly completed.
Cell proliferation not only sustains overall lung growth in the embryo, but also influences lung patterning, since the rate of epithelial proliferation is higher in branching regions (Weaver et al., 2000). One possible result of insufficient epithelial proliferation is decreased branching and cyst formation. The phenotype we observed in SFTPC/Elf5 embryos, where ELF5 specifically inhibited epithelial proliferation in the cysts, is consistent with this concept. Analysis of a lung-specific Elf5 knockout mouse model will be required to determine if ELF5 directly inhibits lung epithelial cell proliferation. The possibility that ELF5 acts as a negative regulator of epithelial proliferation may be tissue type-dependent, however, since mammary epithelial cell proliferation decreases in mice heterozygous for a null Elf5 allele (Zhou et al., 2005).
Increased ELF5 represses distal epithelial cell differentiation
Although distal epithelial expression of ELF5 is high during branching morphogenesis, it is silenced postnatally. One possible explanation for this dynamic expression pattern is that ELF5 negatively regulates distal differentiation. Our results showing that overexpression of ELF5 in the distal epithelium causes decreased expression of Sftpc and the loss of ultrastructural hallmarks of type II cell differentiation are consistent with this possibility. In addition to inhibiting Sftpc expression and type II cell differentiation, microarray analysis revealed that 40 genes normally expressed in differentiated adult type II cells were decreased in SFTPC/Elf5 lungs (Table 1). Two of these genes, Erm (Etv5) and Napsa, have previously been shown to be associated with differentiated type II cells (Brasch et al., 2003; Chuman et al., 1999; Lin et al., 2006; Liu et al., 2003; Ueno et al., 2004). ERM directly binds the Sftpc promoter to drive its transcription (Lin et al., 2006). High ELF5 expression may therefore indirectly inhibit Sftpc expression by reducing Erm expression. This may be occurring in the normal adult airway epithelium, where ELF5 is highly expressed and Erm is absent (Lin et al., 2006). It is also possible that ELF5 may compete with ERM for Ets binding sites on the Sftpc promoter, which would directly inhibit Sftpc transcription. The ETS domain of ELF5 can function as a repressor (Choi and Sinha, 2006), suggesting the possibility that ELF5 may directly inhibit Sftpc transcription. Reduced levels of Napsa have also been associated with defective lung epithelial differentiation in double heterozygous Gata6+/−/Nkx2.1+/− mutant embryos (Zhang et al., 2007). Notably, we found that the effects of ELF5 misexpression on distal lung epithelial differentiation occurred both when the transgene was expressed throughout development and when it was induced only from E16.5 to E18.5. This observation was distinct from the effect of ELF5 misexpression on lung morphogenesis, which was stage-dependent, suggesting that ELF5 disrupts lung epithelial branching and differentiation by affecting different target genes.
In addition to Elf5 (Ese2), the Epithelial-Specific Ets family also includes Ese1 (Elf3), Ese3 (Ehf) and Spdef (Pdef) (Feldman et al., 2003), all of which are expressed in the lung (Kas et al., 2000; Lei et al., 2007; Oettgen et al., 1999; Park et al., 2007; Silverman et al., 2002; Zhou et al., 1998). We found that SFTPC/Elf5 lungs contained increased Ese3 and Spdef levels, suggesting the possibility that ELF5 may control their expression. SPDEF, which is expressed in airway epithelial cells, appears to regulate cell differentiation and gene expression, since misexpression of SPDEF in the airway epithelium induced goblet cell hyperplasia (Park et al., 2007). We saw no evidence of goblet cell hyperplasia in the distal epithelial cysts of SFTPC/Elf5 lungs, indicating that the influence of SPDEF on target gene expression and cell phenotype may be regionally specific. ESE-3 is also expressed in the airway epithelium, especially in serous and mucous glands, (Tugores et al., 2001). Little is known about the function of ESE-3 in the lung, other than that it is required for induction of the squamous differentiation marker SPRR1B in human bronchial epithelial cells (Reddy et al., 2003). In other tissues, however, ESE-3 functions as a context-dependent transcriptional repressor that lies downsteam of MAPK signaling (Tugores et al., 2001), in cellular senescence (Fujikawa et al., 2007), and as a tumor suppresor in prostate cancer (Cangemi et al., 2007). Since ELF5 expression increases in proximal airway epithelium late in gestation, and because ELF5 induced the proximal airway epithelial genes Spdef and Ese3, we asked whether the high levels of ELF5 seen in SFTPC/Elf5 lungs induced the distal epithelial cells to adopt a proximal epithelial cell phenotype. We found, however, that CCSP and FoxJ1, markers of Clara and ciliated cells, respectively, were not induced in the cystic distal epithelium, indicating that ELF5 by itself cannot induce fully differentiated airway epithelial cells.
ELF5 in the maintenance of lung epithelial cell fate
Although a primary role of FGF10 in lung morphogenesis is to act as an epithelial cell chemoattractant (Park et al., 1998), it has also been proposed to maintain epithelial cells as undifferentiated progenitor cells (Norgaard et al., 2003; Nyeng et al., 2007; Ramasamy et al., 2007). Our data showing that ELF5 misexpression represses type II cell maturation and the expression of Sftpc, Erm, and Napsa suggest that Elf5, a target gene of FGF10 (Metzger et al., 2007), may act in this process. If this is the case, the silencing of Elf5 in the distal epithelium that normally occurs in late gestation would be required for normal differentiation to proceed. In support of the concept that ELF5 maintains undifferentiated progenitors, we found that ELF5 overexpression induced p63 and Aqp3, genes that normally mark undifferentiated basal cells of the tracheobronchial epithelium (Avril-Delplanque et al., 2005; Daniely et al., 2004; Liu et al., 2007; Rawlins and Hogan, 2006; Sato et al., 2004). Basal cells of pseudostratified epithelia (e.g. tracheal epithelium) and stratified epithelium (e.g. esophageal epithelia and epidermis) are a source of progenitor cells in these tissues (Daniely et al., 2004; Yang et al., 1998). Basal cells in the adult trachea and bronchi can repopulate the injured epithelium and give rise to fully differentiated cell types, suggesting they behave as stem cells (Hong et al., 2004a; Hong et al., 2004b). The fact that we observed many p63 positive cells in SFTPC/Elf5 lungs suggests the possibility that the dilated cysts may represent a population of progenitor cells. Some reports have suggested that p63 is important for the maintenance of progenitor/stem cells, but this is not fully resolved and is complicated by the existence of various p63 isoforms that can perform distinct functions (Blanpain and Fuchs, 2007; Koster et al., 2007; Koster et al., 2004; Koster et al., 2005; Senoo et al., 2007; Yang et al., 1999).
The identity of the cells constituting the epithelial cysts is unclear, but the fact that they express TTF1 indicates that they are in the respiratory lineage (Zhou et al., 1996). Although our microarray data showed that high expression of ELF5 repressed some genes that were present in adult type II cells, it simultaneously induced others, such as Sftpd, Spink5, and Scgb3a1. It should be noted, however, that these genes are not restricted to type II cells, since they are also expressed in proximal airway epithelium (Crouch et al., 1992; Reynolds et al., 2002; Walden et al., 2002; Wong et al., 1996). These cells, however, are not fully differentiated airway epithelial cells, since they lack markers for type II, Clara, or ciliated cells.
Disruption of the normal program of epithelial differentiation by high levels of ELF5 may activate new downstream targets that allow the cells to adopt a different fate, as evidenced by our observation that Spink4, Guca2a, Tff3 and Dcpp were induced in the cysts of SFTPC/Elf5 lungs. These are genes that are normally expressed in the intestine and not the lung parenchyma. A previous study has shown that hyperactivation of β-catenin transcriptional activity directed to lung epithelial progenitors produced dilated epithelial cysts and suppressed type II cell differentiation (Okubo and Hogan, 2004). Some of the cells in the cysts expressed Spink4, Guca2a, Tff3 and Dcpp, and the authors concluded that these cells had been induced to change fate by a process termed transdetermination. Although we observed that these genes were induced in SFTPC/Elf5 lungs, our microarrays showed no changes in Wnt-related gene expression. Furthermore, Okubo et al. did not report a change in Elf5, indicating that Elf5 is not downstream of WNT signaling. A more recent study by Nyeng et al. showed that FGF10 misexpressed under control of the Pdx1 promoter caused cystic dilation of the branching lung with induced expression of Tff3 (Nyeng et al., 2008). The epithelial cells in these lungs co-expressed markers associated with the early distal lung epithelium (e.g. SP-C and TTF1), but did not show evidence of transdetermination, leading the authors to conclude that excessive FGF10 signaling arrested epithelial cell differentiation and maintained the cells as progenitors. This phenotype is similar, but not identical, to that which we observed in SFTPC/Elf5 mice, which were SP-C negative. Because we have previously shown that Elf5 is a downstream target of FGF10 (Metzger et al., 2007), we speculate that some of the effects of FGF10 misexpression on maintaining epithelial cells in a progenitor state may be due to increased expression of ELF5.
In summary, we have used an inducible transgenic mouse model to show that high levels of ELF5 in the distal lung epithelium disrupt normal lung morphogenesis and epithelial cell differentiation. Our results suggest that ELF5 must be present at precise levels for the proper specification and subsequent differentiation of lung epithelial cells. Our data also suggest that the role of ELF5 in lung epithelial cell differentiation is complex, and may depend on other components in the transcriptional networks regulating the differentiation of individual lung epithelial cell types.
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ACKNOWLEDGEMENTS
This work was supported by NHLBI R01 HL084376 and training grant HL07752. The authors gratefully acknowledge Drs. Jeff Whitsett and Machiko Ikegami for access to the adult type II cell microarray database, Dr. Yan Xu for maintaining the Pulmonary Biology Gene Expression Database, Dave Loudy for advice on immunohistochemistry, and Dr. Jim Bridges for helpful discussion.
Footnotes
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