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J Biol Chem, Vol. 275, Issue 16, 11858-11864, April 21, 2000


Conditional Expression of Fibroblast Growth Factor-7 in the Developing and Mature Lung*

Jay W. Tichelaar, Wei Lu, and Jeffrey A. WhitsettDagger

From the Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio 45229-3039

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation were assessed in transgenic mice in which the human FGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline induced FGF-7 mRNA in respiratory epithelial cells in both Sp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)7-cmv-fgf-7 transgenes survived, and lung morphology was normal. Induction of FGF-7 expression by doxycycline in the Ccsp-rtta × (Teto)7-cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rtta and Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibroblast growth factor-7 (FGF-7)1 is a potent mitogen that enhances cell proliferation in various organs, including the skin, intestine, breast, liver, and lung. In the lung, FGF-7 and the related polypeptide FGF-10 are expressed in mesenchymal cells in distinct temporal and spatial patterns. Fgf-7 expression begins at embryonic day 14.5, being detected throughout the mesenchyme surrounding the developing lung tubules (1, 2). Fgf-10 expression is initiated earlier, at the onset of lung organogenesis, and is restricted to the mesenchyme surrounding the distal tips of the branching tubules (3). FGF-7 and FGF-10 bind with high affinity to the same FGF receptor 2 (FGFR2-IIIb) isoform (4); however, FGF-10 also binds to FGFR1-IIIb (5). The expression of FGFR2 is restricted to the epithelial cells of the developing lung at the onset of lung organogenesis at embryonic day 9.5 and becomes increasingly restricted to peripheral respiratory epithelial cells as lung development progresses (6, 7). The distinct patterns of expression of Fgf-7 and Fgf-10 mRNAs in lung mesenchyme and the complementary expression of Fgfr-2 in respiratory epithelial cells support a role for FGF signaling in lung morphogenesis.

Experimental support for the role of FGF in epithelial/mesenchymal signaling during lung development has been obtained in vitro and in vivo. Ectopic FGF-7 caused cystadenomatoid malformations in the fetal lung, associated with increased chloride-dependent fluid secretion, cell proliferation, and disrupted branching morphogenesis in vivo and in vitro (8, 9). Fgf-7 expression in the fetal lung in vivo was uniformly lethal by 16 days of gestation and was likely related to the massive cyst formation induced by the polypeptide. Conversely, genetic ablation of Fgf-7 did not cause pulmonary abnormalities (10), indicating that this polypeptide was not required for normal lung morphogenesis, but rather may play a role in pulmonary homeostasis following injury.

A primary role for FGF-10 in lung development was demonstrated by the finding that targeted disruption of Fgf-10 resulted in mice lacking lungs and limbs (11, 12). In vitro, lung tubules migrate toward FGF-10-soaked beads, consistent with a chemotactic function for FGF-10 (13). Recently, creation of tetraploid fusion chimeras lacking embryonic expression of Fgfr-2 yielded mice with a lungless and limbless phenotype, similar to that seen in Fgf-10 gene-targeted mice (14).

FGF-7 is also a potent mitogen in the postnatal lung. Intratracheal instillation of FGF-7 caused transient, but marked, epithelial cell hyperplasia of both bronchiolar and alveolar epithelial cells in the lungs of rats and mice (15, 16). Following FGF-7 treatment, cell proliferation peaks in 2-3 days, and lung structure and proliferative activity returns to normal 1-2 weeks after administration. Since FGF-7 has marked pro-mitotic activities in respiratory epithelial cells, its potential utility for prevention or treatment of lung injury has been explored in vivo. Pretreatment of the lung with FGF-7 protects animals from the effects of oxygen-, acid-, bleomycin-, or radiation-induced lung injury (17-20). Additionally, FGF-7 has been shown to be increased in the lung in animal models of lung injury (21), suggesting that FGF-7 has important functions in regulating the response of the lung to injury.

Since Fgf-7 was uniformly fatal in utero when continuously expressed in the fetal lung, animal models that assess the chronic effects of FGF-7 on lung structure and function in vivo have not been developed. In the present work, transgenic mice were designed in which the expression of FGF-7 was placed under the control of a doxycycline-inducible gene control system (22), directed either by the 3.7-kb human SP-C or the 2.3-kb rat ccsp gene promoters. By using this conditional system, fetal lethality observed in previous FGF-7 transgenic models was overcome. Induction of FGF-7 in utero altered branching morphogenesis and caused cystic dilation of the developing lung tubules. In the adult mouse lung, induction of FGF-7 altered gene expression caused widespread alveolar and bronchiolar hyperplasia that was largely reversible.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction and Oocyte Microinjection-- The rtta construct was a gift of Dr. Herman Bujard (ZMBH, Heidelberg, Germany), and the 1-kb rtta coding sequence was placed under the control of the 3.7-kb human SP-C promoter (23) or the 2.3-kb rat ccsp promoter (24) that selectively direct expression of transgenes in respiratory epithelial cells of the lung (Fig. 1). Polyadenylation sequences from SV40 or the human growth hormone gene were used to ensure transcript termination. Plasmid constructs were verified by sequencing and then microinjected into mouse oocytes using standard transgenic procedures.

PCR Genotyping-- Transgenic mice were identified using PCR primers specific for each transgene as follows: 5' primer in SP-C promoter, 5'-GAC ACA TAT AAG ACC CTG GTC A-3'; 5' primer in ccsp promoter, 5'-ACT GCC CAT TGC CCA AAC AC-3'; 3' primer in rtta coding sequence used for genotyping both Sp-c-rtta and Ccsp-rtta mice, 5'-AAA ATC TTG CCA GCT TTC CCC-3'. Primers used for identification of (Teto)7-cmv-fgf-7 transgene are as follows: 5' primer in CMV minimal promoter, 5'-GCC ATC CAC GCT GTT TTG-3'; 3' primer in hFGF-7 coding region, 5'-CAT TTC CCC TCC GTT GTG-3'. Amplification of PCR product for Sp-c-rtta and Ccsp-rtta was performed by denaturation at 94 °C for 5 min and then 30 cycles of amplification at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, followed by a 7-min extension at 72 °C. Detection of (Teto)7-cmv-fgf-7 was identical except the annealing temperature was 54 °C.

Animal Use and Administration of Doxycycline-- Animals were housed under pathogen-free conditions in accordance with institutional guidelines. A 50× doxycycline HCl stock (25 mg/ml in 50% ethanol, Sigma) was freshly prepared prior to each administration of the drug. Oral doxycycline was administered in the drinking water at a final concentration of 0.5 mg/ml and 1% ethanol. Due to the light sensitivity of doxycycline, doxycycline water was replaced three times per week. Animals not receiving doxycycline were treated with vehicle only (1% ethanol final concentration).

RNA Isolation and RT-PCR-- Lung tissue was homogenized in Tri-Zol reagent (Life Technologies, Inc.) and RNA isolated following manufacturer specifications. RT-PCR was used to detect transgene expression and distinguish between endogenous Fgf-7 and the human FGF-7 transgene. Reverse transcription was carried out on 1 µg of total lung RNA in the presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.5 mM each dNTP, and 25 ng/µl oligo(dT) at 42 °C for 1 h. PCR primers for beta -actin are as follows: beta -actin primer 1, 5'-GTG GGC CGC TCT AGG CAC CAA-3'; beta -actin primer 2, 5'-CTC TTT GAT GTC ACG CAG GAT TTC-3'; for human FGF-7: hFGF-7 primer 1, 5'-ATA TCA TGG AAA TCA GGA CA-3'; hFGF-7 primer 2, 5'-CAT TTC CCC TCC GTT GTG-3'. PCR conditions for hFGF-7 were 94 °C denaturation for 5 min, followed by 25 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, followed by a 7-min extension at 72 °C. PCR conditions for beta -actin were similar, but the annealing temperature was 59 °C. All reactions were run in duplicate with a negative control reaction lacking reverse transcriptase enzyme. No amplification was seen in reactions lacking reverse transcriptase (data not shown).

Lung Histology and Immunohistochemistry-- Lungs were inflation fixed using 4% paraformaldehyde at 25 cm of pressure and then allowed to fix overnight at 4 °C, washed with phosphate-buffered saline (PBS), dehydrated through a graded series of ethanols, and processed for paraffin embedding. 5-µm sections were loaded onto polylysine slides for immunostaining. Antibodies and procedures for immunostaining of TTF-1 and pro-SP-C have been previously described (25). The AQP5 polyclonal antibody, a gift from Dr. Anil Menon (University of Cincinnati), was used at a 1:1000 dilution and has been previously characterized (26). For immunohistochemical detection of BrdUrd, animals were injected with 0.1 ml of BrdUrd labeling reagent (Zymed Laboratories Inc.) per 100 × g of body weight 2 h prior to sacrifice. BrdUrd incorporated into DNA was detected using anti-BrdUrd monoclonal antibody and a BrdUrd staining kit (Zymed Laboratories Inc.). Volume of inflated lungs was determined after overnight fixation and an initial PBS wash. Inflated lungs were dissected from surrounding tissue and weighed by immersion in PBS.

In Situ Hybridization-- Paraffin-embedded lung tissue was cut at 5 µm and loaded onto silanized slides. Sense and antisense riboprobes were generated from a murine Fgf-7 cDNA or rtta cDNA cloned into pGEM3z and transcribed in vitro with a Riboprobe transcription kit (Promega). Conditions and solutions for hybridization are essentially as described (27). For the FGF-7 sense and antisense probes, hybridization was carried out overnight at 42 °C and washed under low stringency conditions. For the rtta sense and antisense probes, hybridization was carried out overnight at 55 °C and washed under high stringency conditions. Slides were dipped in Kodak NTB2 emulsion and exposed for 3-7 days. Slides were developed with Kodak D19 developer following the manufacturer's protocol.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of rtTA and (Teto)7-cmv-fgf-7 Transgenic Mice-- Transgenic mice were produced bearing the reverse tetracycline-responsive transactivator (rtta) fusion protein under control of the 3.7-kb human SP-C gene promoter or the 2.3-kb rat ccsp gene promoter (Fig. 1), hereafter referred to as Sp-c or Ccsp "activator" mice. The resulting founders were bred to establish permanent lines, screening for expression of the rtta transgene by RT-PCR and Northern blotting. Transgene expression was detected in only 1 of 15 Sp-c-rtta and 1 of 7 Ccsp-rtta founders (data not shown). Separate transgenic mouse lines were generated by microinjection of a (Teto)7-cmv-fgf-7 transgene (Fig. 1A), and two separate mouse lines bearing the target transgene were established. The (Teto)7-cmv-fgf-7 transgene consists of seven copies of the tet operator DNA binding sequence linked to a minimal CMV promoter (22), the human FGF-7 cDNA, and SV40 polyadenylation signals. Transgenic mice bearing either the Sp-c-rtta, Ccsp-rtta, or (Teto)7-cmv-fgf-7 transgenes had no lung or other tissue pathologies and survived normally in the vivarium. To obtain transgenic mice in which FGF-7 expression was regulated by administration of doxycycline, Sp-c or Ccsp activator mice were bred to (Teto)7-cmv-fgf-7 target mice producing bitransgenic progeny (Fig. 1B).


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  		Fig. 1.   Constructs used to generate doxycycline-regulatable transgene expression in the lung. A, the Sp-c-rtta transgene consists of the 3.7-kb human SP-C promoter, 1.0-kb rtta coding sequence, and 0.45-kb SV40 polyadenylation signal. The Ccsp-rtta transgene consists of the 2.3-kb rat ccsp promoter, 1.0-kb rtta coding sequence, and a 2.0-kb fragment from the human growth hormone gene containing introns and a polyadenylation signal. The (Teto)7-cmv-fgf-7 transgene consists of seven copies of the tet operator, a CMV minimal promoter, the human FGF-7 coding sequence, and SV40 polyadenylation signal. B, single transgenic mice bearing either the Ccsp-rtta or Sp-c-rtta transgenes were bred to single transgenic mice bearing the (Teto)7-cmv-fgf-7 transgene to generate double transgenic progeny.

Induction of FGF-7 Expression by Doxycycline in Utero-- To determine the effects of inducing FGF-7 expression in utero utilizing the Ccsp-rtta activator mice, double transgenic Ccsp-rtta × (Teto)7-cmv-fgf-7 mice were generated. Fetal pups were obtained at embryonic day 17 after 7 days of doxycycline administration to the dam. Histological examination of the lungs from doxycycline treated Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic pups revealed marked cystadenomatoid malformation similar to, but less severe than, that induced by FGF-7 previously (Fig. 2B) (8). Lung histology in littermates bearing only the (Teto)7-cmv-fgf-7 transgene was normal (Fig. 2A).


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  		Fig. 2.   Histology and in situ hybridization of fetal lung tissue. Lung tissue from day 17 post-conception fetal mice (A and B) or day 18.5 post-conception fetal mice (C-H) were stained with hematoxylin and eosin for histology (A-E) or hybridized with a riboprobe to detect FGF-7 mRNA (F-H). Lung histology in a (Teto)7-cmv-fgf-7 single transgenic pup (A) and a Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic littermate (B) whose dam was doxycycline-treated for 7 days (post-conception days 10-17). Normal lung histology was observed in single transgenic (Teto)7-cmv-fgf-7 pups at day 18.5 post-conception following 1 day of doxycycline (C). D represents lung from a Sp-c-rtta × (Teto)7-cmv-fgf-7 bitransgenic pup without doxycycline, demonstrating marked hyperplasia and disruption of branching. E represents lung histology from a Sp-c-rtta × (Teto)7-cmv-fgf-7 bitransgenic pup exposed to doxycycline for 1 day. FGF-7 mRNA was not detected by in situ hybridization in a (Teto)7-cmv-fgf-7 single transgenic pup treated with doxycycline for 1 day (F). FGF-7 mRNA was detected in epithelial cells of an age-matched Sp-c-rtta × (Teto)7-cmv-fgf-7 bitransgenic pup without doxycycline (G), or 1 day after exposure to doxycycline (H). Figure is representative of at least four animals per genotype.

Cystadenomatoid malformations were detected in the lungs of Sp-c-rtta × (Teto)7-cmv-fgf-7 mice in the absence of doxycycline (Fig. 2D). Consistent with this finding, human FGF-7 mRNA was detected in the lungs of these double transgenic mice in the absence of doxycycline but was increased 24 h after administration of doxycycline to the dam's drinking water (Fig. 2, G and H). Transgenic FGF-7 mRNA was not detected in single transgenic (Teto)7-cmv-fgf-7 (Fig. 2F) or Sp-c-rtta (not shown) transgenic mice in the presence or absence of doxycycline. Without doxycycline, double transgenic Sp-c-rtta × (Teto)7-cmv-fgf-7 pups survived until birth but died in the immediate postnatal period, likely relating to the abnormalities in lung structure and function. Because of the expression of FGF-7 mRNA in Sp-c-rtta × (Teto)7-cmv-fgf-7 double transgenic mice in the absence of doxycycline, Ccsp-rtta activator mice were used for subsequent studies.

Induction of FGF-7 Expression in the Postnatal Lung-- In the absence of doxycycline, double transgenic Ccsp-rtta × (Teto)7-cmv-fgf-7 mice survived postnatally, and no abnormalities in lung morphology were observed (Fig. 3B). Administration of doxycycline to the drinking water of Ccsp-rtta × (Teto)7-cmv-fgf-7 mice caused marked respiratory epithelial hyperplasia in the lungs of adult mice (Fig. 3C). No pulmonary abnormalities were observed in Ccsp-rtta or (Teto)7-cmv-fgf-7 single transgenic mice treated with doxycycline (Fig. 3A) or in bitransgenic mice that did not receive doxycycline (Fig. 3B). Hyperplasia of the alveolar epithelium was first observed after 4 days of treatment with doxycycline (not shown) and was most pronounced near the pleural surface and surrounding conducting airways. Seven days after treatment with doxycycline, epithelial cell hyperplasia became more widespread. The bronchial and bronchiolar epithelium was thickened and was often lined by a pseudostratified rather than columnar epithelium (Fig. 3C). Epithelial cell hyperplasia was increasingly widespread 14 or 21 days after exposure to doxycycline (Fig. 3, E and G). The lungs of Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice contained increased numbers of alveolar macrophages that increased with continued doxycycline exposure (Fig. 3D). Additionally, the interstitial space between conducting airways, blood vessels, and lung parenchyma was typically widened, becoming more pronounced with increasing duration of doxycycline administration (Fig. 3, C and E). There was no evidence of fibrosis or collagen deposition as assessed by trichrome staining (data not shown). Adult Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice treated with doxycycline for 21 days had dramatically enlarged lungs, although their body weights were less than untreated bitransgenic or doxycyclin-treated (Teto)7-cmv-fgf-7 single transgenic littermates (Fig. 4). Continued treatment of bitransgenic mice with doxycycline caused respiratory distress and was generally lethal by 21 days.


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  		Fig. 3.   Effects of FGF-7 expression in lungs from Ccsp-rtta activated mice. Normal histology was observed in lungs from adult (Teto)7-cmv-fgf-7 single transgenic mice treated with doxycycline for 7 days (A) and in untreated Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice (B). Treatment of Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic littermates with doxycycline for 7 days (C and D) caused marked bronchial and alveolar epithelial cell hyperplasia. Widened interstitial spaces were noted along conducting airways (arrow, C), and macrophage infiltrates were extensive (arrowheads, D). Extensive hyperplasia was noted in Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice following 21 days of doxycycline treatment (E and G). Hyperplasia and macrophage infiltrate resolved in Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic littermates treated for 21 days with doxycycline, followed by 14 days of recovery without doxycycline (F and H). Figure is representative of at least four animals for each genotype.


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  		Fig. 4.   Lungs from Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice. Lungs were inflation fixed at 25 cm of pressure from (Teto)7-cmv-fgf-7 single transgenic mice treated with doxycycline for 21 days (tetO-FGF-7, 21 d Dox), untreated Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice (Dbl. Tg., - Dox), or Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice treated with doxycycline for 21 days. After overnight fixation PBS displacement was used to determine lung volume (graph).

Doxycycline Increased FGF-7 mRNA in Ccsp-rtta × (Teto)7-cmv-fgf-7 Double Transgenic Mice-- In situ hybridization with a mouse Fgf-7 riboprobe was used to detect FGF-7 mRNA in lung tissues of transgenic mice. In the absence of doxycycline, FGF-7 mRNA was undetectable in the lungs of Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice and in single transgenic littermates treated with doxycycline (Fig. 5, A and B). Treatment of Ccsp-rtta × (Teto)7-cmv-fgf-7 mice for 7 days with doxycycline increased FGF-7 mRNA in conducting airway and alveolar epithelial cells (Fig. 5C).


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  		Fig. 5.   Localization of hFGF-7 and rtTA mRNA in adult Ccsp-rtta × (Teto) 7-cmv-fgf-7 lung. FGF-7 mRNA was not detected by in situ hybridization in lungs from (Teto)7-cmv-fgf-7 single transgenic mice treated with doxycycline for 7 days (A) or untreated CCSP-rtTA × (Teto)7-cmv-fgf-7 bitransgenic mice (B). FGF-7 mRNA was readily detected in lungs from CCSP-rtTA × (Teto)7-cmv-fgf-7 bitransgenic mice treated with doxycycline for 7 days, the mRNA being detected in alveolar and bronchial/bronchiolar epithelium (C). No hybridization signal was detected in Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice treated with doxycycline for 7 days when a sense riboprobe was used (D). In situ hybridization with a rtta riboprobe detected scattered signal throughout the alveolar epithelium of Ccsp-rtta single transgenic mice treated with doxycycline for 7 days (E) or in untreated Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice (F). Strong hybridization was detected in alveolar and bronchial/bronchiolar epithelium of Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice treated with doxycycline for 7 days (G). No signal was detected in Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice when a sense probe was used (H).

In situ hybridization was used to identify cells expressing the rtta transgene under the control of the rat ccsp promoter. In Ccsp-rtta transgenic mice, rtTA mRNA was readily detected in bronchial and type II epithelial cells (Fig. 5E) in a pattern nearly identical to that seen in Ccsp-rtta × (Teto)7-cmv-fgf-7 double transgenic mice in the absence of doxycycline (Fig. 5F). Treatment of double transgenic mice for 7 days with doxycycline increased Rtta mRNA in respiratory epithelial cells in a pattern similar to that of the Fgf-7 transgene (Fig. 5G). Thus, treatment of Ccsp-rtta × (Teto)7-cmv-fgf-7 double transgenic mice with doxycycline induced both FGF-7 and Rtta mRNAs.

FGF-7 Enhanced BrdUrd Labeling and Altered Epithelial Cell Markers in Vivo-- Incorporation of BrdUrd into DNA was used to estimate cell proliferation in the transgenic mice. Although no changes in BrdUrd uptake were detected in the lungs of Ccsp-rtta × (Teto)7-cmv-fgf-7 mice 24 h after doxycycline (not shown), increased BrdUrd labeling of epithelial cells was noted after 4 days of doxycycline (Fig. 6B), the number of BrdUrd-labeled cells increased further after 7 days (Fig. 6C). BrdUrd staining was detected in respiratory epithelial cells in the lung periphery, although BrdUrd-labeled cells were occasionally detected in bronchial and bronchiolar epithelial cells. Administration of doxycycline to double transgenic Ccsp-rtta × (Teto)7-cmv-fgf-7 mice increased the intensity of staining for TTF-1 (Fig. 7, C and D) and pro-SP-C (Fig. 7, A and B), the latter a specific marker for type II epithelial cells in the lung periphery. Both pro-SP-C and TTF-1 were readily detectable in the lungs of bitransgenic mice treated with doxycycline at dilutions of antibody at which little or no signal was detected in double transgenic littermates that did not receive doxycycline (Fig. 7, C and D). Respiratory epithelial markers SP-B, pro-SP-B, and HNF-3beta were also increased in doxycycline-treated bitransgenic animals compared with untreated bitransgenic littermates (data not shown). Additionally, immunostaining for the CCSP protein, a Clara cell marker, was maintained in the hyperplastic bronchial and bronchiolar epithelium and was not detected in regions of alveolar hyperplasia (data not shown). Aquaporin 5 (AQP5) and proSP-C staining was increased 7 days after treatment with doxycycline (Fig. 7, B and F), AQP5 being detected in cuboidal and squamous epithelial cells in the lung periphery as well as bronchiolar epithelial cells. The hyperplastic epithelial cells stained heavily for AQP5 on apical membranes.


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  		Fig. 6.   Immunohistochemical staining to detect BrdUrd incorporation. Staining for BrdUrd was detected infrequently in lungs of untreated Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice (arrow, A). Increased staining for BrdUrd in alveolar epithelial cells was apparent following 4 days of doxycycline treatment (B) and was further increased in alveolar epithelial cells following 7 (C) or 21 days (D) of doxycycline treatment. BrdUrd staining was also detected in pleural cells (arrow, C) and macrophages (arrowheads, D) following doxycycline treatment. Figure is representative of at least two animals for each time point.


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  		Fig. 7.   Immunohistochemical staining for proSP-C, TTF-1, and AQP5. Staining for pro-SP-C (A and B), TTF-1 (C and D) and AQP5 (E and F) in adult lung tissue of Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice without doxycycline treatment (A, C, and E) or following 7 days of doxycycline treatment (B, D, and F). Staining for pro-SP-C, a marker for type II alveolar epithelial cells, and for TTF-1 was increased in Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice treated with doxycycline (Dox) for 7 days compared with untreated littermates. Staining for AQP5, normally found in squamous type I alveolar epithelial cells, was abundant in cuboidal type II alveolar epithelial cells and in bronchial epithelial cells following treatment of Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice with doxycycline for 7 days. b, bronchi. Figure is representative of at least two mice from each genotype.

Kinetics of Transgene Induction and Reversible Expression-- Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice were exposed to doxycycline in the drinking water for 1-21 days. Transgene expression was assessed using primers that amplified the human FGF-7 cDNA but not the mouse Fgf-7 cDNA. In the absence of doxycycline, human FGF-7 mRNA was undetectable (Fig. 8A). In contrast, human FGF-7 mRNA was readily detected after 24 h of doxycycline and increased during the chronic administration of doxycycline. Mice that had been treated with doxycycline for 7 days were provided water without doxycycline and studied for 1-7 days thereafter. Human FGF-7 mRNA was decreased 1 day after removal of doxycycline and thereafter (Fig. 8B). Lung histology was assessed in Ccsp-rtta × (Teto)7-cmv-fgf-7 double transgenic mice after cessation of doxycycline treatment. Respiratory epithelial hyperplasia was substantially reduced 7 days after cessation of doxycycline (not shown) and was nearly resolved 14 days after cessation of doxycycline (Fig. 3, F and H), with nearly complete restoration of normal lung parenchymal morphology.


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  		Fig. 8.   Reversibility of human FGF-7 expression in Ccsp-rtta × (Teto) 7-cmv-fgf-7 bitransgenic mice. Specific primers were used to detect the human FGF-7 mRNA by RT-PCR in total lung mRNA obtained from adult Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice. A, bitransgenic mice were treated with doxycycline for 0, 1, 4, 7, 14, or 21 days as indicated above each lane. Control reactions with beta -actin primers are shown in the bottom panel. B, bitransgenic mice were treated with doxycycline for 7 days, 7 days followed by 1 day without doxycycline (lanes 7/1), 7 days followed by 2 days without doxycycline (lanes 7/2), or 7 days followed by 7 days without doxycycline (lanes 7/7). Control reactions with beta -actin primers are shown in the bottom panel. No amplification was seen when reverse transcriptase was omitted from the RT reaction (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have established a system for conditional expression of FGF-7 in the lungs of transgenic mice, overcoming the prenatal lethality associated with expression of FGF-7 in the developing lung in vivo. Treatment of fetal or adult mice with doxycycline caused a rapid induction of FGF-7 mRNA, resulting in cystadenomatoid malformation in fetal lung and epithelial hyperplasia in adult lung. In the adult lung, FGF-7 increased BrdUrd uptake and staining for pro-SP-C and TTF-1, consistent with proliferation and differentiation of type II epithelial cells. The effects of FGF-7 on cellular hyperplasia and targeted gene expression were largely reversed after removal of doxycycline. The Ccsp-rtta and Sp-c-rtta activator mice will likely be useful in generating mouse models in which diverse biologically active molecules are selectively expressed under control of exogenous doxycycline in developing or mature lung.

Postnatal Effects of FGF-7-- FGF-7 caused respiratory epithelial hyperplasia and increased staining for a number of type II epithelial markers including TTF-1, HNF-3beta , proSP-C, and SP-B. Epithelial hyperplasia was first seen in Ccsp-rtta × (Teto)7-cmv-fgf-7 bitransgenic mice 4 days after doxycycline treatment and persisted for up to 3 weeks. In contrast, FGF-7 caused epithelial cell proliferation and hyperplasia 3 days after intratracheal administration that resolved within 7 days (15). Whereas the effects of intratracheal FGF-7 were noted primarily in the alveolar region, hyperplasia was also noted in the conducting airways after doxycycline exposure in our model, demonstrating that conducting airway epithelial cells are also responsive to the growth factor. Pleural thickening, associated with increased BrdUrd uptake, was also observed after chronic expression of FGF-7. The present observations are consistent with the finding that antibodies to FGF-7 blocked stimulatory effects of pleural lavage fluid from asbestos-treated rats on pleural mesothelial cells (28).

The interstitial space surrounding conducting airways and blood vessels was enlarged in Ccsp-rtta × (Teto)7-cmv-fgf-7 mice treated with doxycycline although the interstitium was not fibrotic. The lack of cells or extracellular matrix accumulation suggests that the increase in interstitial space may result from fluid accumulation. This possibility is supported by a dramatic increase and redistribution of AQP5 from its normal expression in type I alveolar epithelial cells to the hyperplastic alveolar epithelial cells found in Ccsp-rtta × (Teto)7-cmv-fgf-7 double transgenic mice. Previous studies demonstrated that FGF-7 regulates fluid balance in the fetal lung (8, 9, 29), and the present findings suggest that FGF-7 regulates AQP5 expression, an epithelial water channel. The increased expression of AQP5 in vivo is in contrast to a previous report demonstrating that FGF-7 inhibited Aqp5 expression during in vitro culture of alveolar type II cells (30). The distinct effects of FGF-7 on Aqp5 expression in the two systems may represent different responses of alveolar epithelial cells in vitro versus in vivo. Pulmonary edema is common in lung injury, and increased FGF-7 may be involved in resolution of edema. Consistent with this hypothesis, it is known that FGF-7 decreases pulmonary edema (31), probably by increasing alveolar liquid clearance (32). In vitro, or following intratracheal doses, FGF-7 also regulates other mediators of fluid balance such as the epithelial sodium channel, the sodium/potassium-ATPase (Na+/K+-ATPase), and the epithelial chloride channel ClC-2 (33-36).

FGF-7 caused a marked increase in the number and BrdUrd labeling of alveolar macrophages. It is unclear whether this represents a direct effect of FGF-7 on macrophage recruitment or proliferation of alveolar precursors or is a response to the epithelial cell hyperplasia and remodeling of lung parenchyma.

Chronic stimulation of FGF-7 in the lung caused severe hyperplasia that resembled a pre-cancerous human lesion, termed alveolar adenomatous hyperplasia (37). Withdrawal of doxycycline caused nearly complete remodeling of the lung, although mild epithelial cell hyperplasia was observed 14 days after cessation of doxycycline. Resolution of FGF-7-induced hyperplasia is thought to be mediated by apoptosis of type II cells and differentiation of type II cells into type I cells (38). After removal of doxycycline, the normal pattern of immunostaining of APQ5 in squamous epithelial cells was restored, consistent with differentiation of type II cells into type I cells. The reversible hyperplasia seen in our model agrees with recent transgenic models of neoplasia (39-41) and suggests that inappropriate expression of FGF-7 was not oncogenic.

Induction of the Ccsp-rtta Transgene by FGF-7-- The Rtta mRNA was detected in type II alveolar epithelial cells as well as in conducting airway epithelial cells in Ccsp-rtta single transgenic mice. The presence of Rtta mRNA in type II cells was somewhat surprising since the ccsp promoter typically directs transgene expression to non-ciliated epithelial cells in the conducting airway. We have previously observed type II cell expression using the rat ccsp promoter in transgenic mice, perhaps reflecting species differences or positional effects that influence the sites of expression.2 FGF-7 caused proliferation of alveolar epithelial cells expressing the (Teto)7-cmv-fgf-7 transgene and also increased staining for TTF-1 within these cells. It is therefore possible that FGF-7 increased Ttf-1 expression that, in turn, may have influenced the level of activity of the rat ccsp promoter per se (42), increasing levels of Rtta that may further induce the activity of the (Teto)7-cmv-fgf-7 transgene.

Cystadenomatoid Malformation Induced by FGF-7 in Fetal Lung-- Conditional expression of FGF-7 in the fetal lung caused cystadenomatoid malformation in vivo, consistent with previous in vitro and in vivo findings (8, 9). The severity of malformation induced by Ccsp-rtta as compared with Sp-c-rtta may be related to the timing and level of expression, the SP-C promoter being active earlier in gestation (27). Although lung abnormalities were not observed in Ccsp-rtta-activated mice in the absence of doxycycline, cystic abnormalities were seen in the Sp-c-rtta-activated mice in the absence of doxycycline. The ability of low levels of FGF-7 to induce a cystic phenotype is consistent with the potent effect of FGF-7 on respiratory epithelial cells (43). FGF-7 expression was induced in the fetal lung of Sp-c-rtta × (Teto)7-cmv-fgf-7 bitransgenic pups by the addition of doxycycline to the drinking water of the dam, indicating that the system remains inducible and of potential utility for study of lung morphogenesis and function.

Distinct Effects of FGF-7 and FGF-10-- FGF-7 and FGF-10 both bind to the FGFR2(IIIb) receptor found on respiratory epithelial cells, predicting similar phenotypes following induction of these growth factors in the lung. However, effects of FGF-7 on lung morphogenesis are distinct from those caused by FGF-10,3 suggesting that signaling by these closely related FGF polypeptides utilizes distinct pathways or modifiers. Recently it was shown that FGF-10, but not FGF-7, binds to FGFR1(IIIb) (5), a receptor present in pulmonary mesenchyme (1, 7). Whether the distinct effects of FGF-7 and FGF-10 on lung morphogenesis are influenced by FGF-10-mediated pathways in mesenchyme remains to be elucidated. Heparan sulfate proteoglycans influence signaling through FGFRs (44), and the patterns of heparan sulfate expression and the extent of their glycosylation vary developmentally (45). Thus, the differences in phenotype in FGF-7 and FGF-10 expressing mice may reflect differences in receptor activation by each ligand related to developmental changes in heparan sulfate proteoglycans or other modifiers of FGF-signaling pathways in target cells.

Lung-specific, Doxycycline-inducible Transgenic Mouse Models-- Generation of Ccsp-rtta × (Teto)7-cmv-fgf-7 double transgenic mice provides a tightly regulated system for studying the effects of FGF-7 in the respiratory epithelium. The utility of the Ccsp system is evidenced by the finding that the human FGF-7 transgene was not detected in unstimulated Ccsp-rtta × (Teto)7-cmv-fgf-7 mouse lung and is further supported by studies using (tetO)7-CMV-luciferase mice to assay levels of Ccsp-rtta regulated transgene induction.4 Transgene expression was not detected in tissues other than lung, and cell proliferation was not increased in the livers of Ccsp-rtta × (Teto)7-cmv-fgf-7 mice treated with doxycycline (data not shown), indicating that the activity of FGF-7 was restricted to the lung. The present findings support previous studies demonstrating doxycycline-regulated expression of IL-11 in mice co-injected with both Ccsp-rtta and (tetO)7-CMV-IL-11 transgenes (46). In those studies, inducible expression of IL-11 and pulmonary abnormalities were observed following doxycycline treatment, although IL-11 was detected in the absence of doxycycline. Since Sp-c-rtta and Ccsp-rtta were generated as independent activator lines, these mice can be bred to various "target" mice conferring conditional expression of exogenous genes in the lung.

    ACKNOWLEDGEMENTS

We thank Drs. P. Ray and J. Elias for helpful discussion and Dr. H. Bujard for the availability of the rtTA and (tetO)7CMV plasmids.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL-56387 and HL-41496 and the Cystic Fibrosis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-636-4830; Fax: 513-636-7868; E-mail: jeff.whitsett@chmcc.org.

2 J. W. Tichelaar, S. Wert, and J. A. Whitsett, unpublished observations.

3 J. C. Clark, J. W. Tichelaar, N. Itoh, A.-K. T. Perl, S. E. Wert, M. T. Stahlman, and J. A. Whitsett, unpublished observations.

4 A.-K. T. Perl, J. W. Tichelaar, and J. A. Whitsett, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: FGF-7, fibroblast growth factor-7; BrdUrd, 5-bromodeoxyuridine; SP-C, surfactant protein-C; CCSP, Clara cell secretory protein; FGFR, FGF receptor; kb, kilobase pair; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; CMV, cytomegalovirus; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Finch, P. W., Cunha, G. R., Rubin, J. S., Wong, J., and Ron, D. (1995) Dev. Dyn. 203, 223-240[Medline] [Order article via Infotrieve]
2. Mason, I. J., Fuller-Pace, F., Smith, R., and Dickson, C. (1994) Mech. Dev. 45, 15-30[CrossRef][Medline] [Order article via Infotrieve]
3. Bellusci, S., Grindley, J., Emoto, H., Itoh, N., and Hogan, B. L. M. (1997) Development 124, 4867-4878[Abstract]
4. Igarashi, M., Finch, P. W., and Aaronson, S. A. (1998) J. Biol. Chem. 273, 13230-13235[Abstract/Free Full Text]
5. Luo, Y., Lu, W., Mohamedali, K. A., Jang, J. H., Jones, R. B., Gabriel, J. L., Kan, M., and McKeehan, W. L. (1998) Biochemistry 37, 16506-16515[CrossRef][Medline] [Order article via Infotrieve]
6. Peters, K. G., Werner, S., Chen, G., and Williams, L. T. (1992) Development 114, 233-243[Abstract]
7. Orr-Urtreger, A., Givol, D., Yayon, A., Yarden, Y., and Lonai, P. (1991) Development 113, 1419-1434[Abstract]
8. Simonet, W. S., DeRose, M. L., Bucay, N., Nguyen, H. Q., Wert, S. E., Zhou, L., Ulich, T. R., Thomason, A., Danilenko, D. M., and Whitsett, J. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12461-12465[Abstract/Free Full Text]
9. Zhou, L., Graeff, R. W., McCray, P. B., Jr., Simonet, W. S., and Whitsett, J. A. (1996) Am. J. Physiol. 271, L987-L994[Abstract/Free Full Text]
10. Guo, L., Degenstein, L., and Fuchs, E. (1996) Genes Dev. 10, 165-175[Abstract/Free Full Text]
11. Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N., and Kato, S. (1999) Nat. Genet. 21, 138-141[CrossRef][Medline] [Order article via Infotrieve]
12. Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B. D., Tarpley, J. E., DeRose, M., and Simonet, W. S. (1998) Genes Dev. 12, 3156-3161[Abstract/Free Full Text]
13. Park, W. Y., Miranda, B., Lebeche, D., Hashimoto, G., and Cardoso, W. V. (1998) Dev. Biol. 201, 125-134[CrossRef][Medline] [Order article via Infotrieve]
14. Arman, E., Haffner-Krausz, R., Gorivodsky, M., and Lonai, P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11895-11899[Abstract/Free Full Text]
15. Ulich, T. R., Yi, E. S., Longmuir, K., Yin, S., Biltz, R., Morris, C. F., Housley, R. M., and Pierce, G. F. (1994) J. Clin. Invest. 93, 1298-1306
16. Zsengeller, Z. K., Halbert, C., Miller, A. D., Wert, S. E., Whitsett, J. A., and Bachurski, C. J. (1999) Hum. Gene Ther. 10, 341-353[CrossRef][Medline] [Order article via Infotrieve]
17. Panos, R. J., Bak, P. M., Simonet, W. S., Rubin, J. S., and Smith, L. J. (1995) J. Clin. Invest. 96, 2026-2033
18. Yi, E. S., Williams, S. T., Lee, H., Malicki, D. M., Chin, E. M., Yin, S., Tarpley, J., and Ulich, T. R. (1996) Am. J. Pathol. 149, 1963-1970[Abstract]
19. Deterding, R. R., Havill, A. M., Yano, T., Middleton, S. C., Jacoby, C. R., Shannon, J. M., Simonet, W. S., and Mason, R. J. (1997) Proc. Assoc. Am. Physicians 109, 254-268[Medline] [Order article via Infotrieve]
20. Yano, T., Deterding, R. R., Simonet, W. S., Shannon, J. M., and Mason, R. J. (1996) Am. J. Respir. Cell Mol. Biol. 15, 433-442[Abstract]
21. Charafeddine, L., D'Angio, C. T., Richards, J. L., Stripp, B. R., Finkelstein, J. N., Orlowski, C. C., LoMonaco, M. B., Paxhia, A., and Ryan, R. M. (1999) Am. J. Physiol. 276, L105-L113[Abstract/Free Full Text]
22. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551[Abstract/Free Full Text]
23. Glasser, S. W., Korfhagen, T. R., Wert, S. E., Bruno, M. D., McWilliams, K. M., Vorbroker, D. K., and Whitsett, J. A. (1991) Am. J. Physiol. 261, L349-L356[Abstract/Free Full Text]
24. Stripp, B. R., Sawaya, P. L., Luse, D. S., Wikenheiser, K. A., Wert, S. E., Huffman, J. A., Lattier, D. L., Singh, G., Katyal, S. L., and Whitsett, J. A. (1992) J. Biol. Chem. 267, 14703-14712[Abstract/Free Full Text]
25. Zhou, L., Lim, L., Costa, R. H., and Whitsett, J. A. (1996) J. Histochem. Cytochem. 44, 1183-1193[Abstract]
26. Krane, C. M., Towne, J. E., and Menon, A. G. (1999) Mamm. Genome 10, 498-505[CrossRef][Medline] [Order article via Infotrieve]
27. Wert, S. E., Glasser, S. W., Korfhagen, T. R., and Whitsett, J. A. (1993) Dev. Biol. 156, 426-443[CrossRef][Medline] [Order article via Infotrieve]
28. Adamson, I., Prieditis, H., and Young, L. (1997) Am. J. Respir. Cell Mol. Biol. 16, 650-656[Abstract]
29. Graeff, R. W., Wang, G., and McCray, P. B., Jr. (1999) Pediatr. Res. 46, 523-529[Medline] [Order article via Infotrieve]
30. Borok, Z., Lubman, R. L., Danto, S. I., Zhang, X. L., Zabski, S. M., King, L. S., Lee, D. M., Agre, P., and Crandall, E. D. (1998) Am. J. Respir. Cell Mol. Biol. 18, 554-561[Abstract/Free Full Text]
31. Yi, E. S., Salgado, M., Williams, S., Kim, S. J., Masliah, E., Yin, S., and Ulich, T. R. (1998) Inflammation 22, 315-325[CrossRef][Medline] [Order article via Infotrieve]
32. Wang, Y., Folkesson, H., Jayr, C., Ware, L., and Matthay, M. (1999) J. Appl. Physiol. 87, 1852-1860[Abstract/Free Full Text]
33. Guery, B. P., Mason, C. M., Dobard, E. P., Beaucaire, G., Summer, W. R., and Nelson, S. (1997) Am. J. Respir. Crit. Care Med. 155, 1777-1784[Abstract]
34. Borok, Z., Danto, S. I., Dimen, L. L., Zhang, X. L., and Lubman, R. L. (1998) Am. J. Physiol. 274, L149-L158
35. Blaisdell, C. J., Pellettieri, J. P., Loughlin, C. E., Chu, S., and Zeitlin, P. L. (1999) Am. J. Respir. Cell Mol. Biol. 20, 842-847[Abstract/Free Full Text]
36. Borok, Z., Mihyu, S., Fernandes, V. F., Zhang, X. L., Kim, K. J., and Lubman, R. L. (1999) Am. J. Physiol. 276, C1352-C1360
37. Colby, T. V. (1999) in Lung Tumors: Fundamental Biology and Clinical Management (Brambilla, C. , and Brambilla, E., eds), Vol. 124 , pp. 61-87, Marcel Dekker, Inc., New York
38. Fehrenbach, H., Kasper, M., Tschernig, T., Pan, T., Schuh, D., Shannon, J. M., Muller, M., and Mason, R. J. (1999) Eur. Respir. J. 14, 534-544[Abstract]
39. Xie, W., Chow, L. T., Paterson, A. J., Chin, E., and Kudlow, J. E. (1999) Oncogene 18, 3593-3607[CrossRef][Medline] [Order article via Infotrieve]
40. Chin, L., Tam, A., Pomerantz, J., Wong, M., Holash, J., Bardeesy, N., Shen, Q., O'Hagan, R., Pantginis, J., Zhou, H., Horner, J. W., II, Cordon-Cardo, C., Yancopoulos, G. D., and DePinho, R. A. (1999) Nature 400, 468-472[CrossRef][Medline] [Order article via Infotrieve]
41. Felsher, D. W., and Bishop, J. M. (1999) Mol. Cell 4, 199-207[CrossRef][Medline] [Order article via Infotrieve]
42. Zhang, L., Whitsett, J. A., and Stripp, B. R. (1997) Biochim. Biophys. Acta 1350, 359-367[Medline] [Order article via Infotrieve]
43. Deterding, R. R., Jacoby, C. R., and Shannon, J. M. (1996) Am. J. Physiol. 271, L495-L505[Abstract/Free Full Text]
44. Kan, M., Wang, F., Kan, M., To, B., Gabriel, J. L., and McKeehan, W. L. (1996) J. Biol. Chem. 271, 26143-26148[Abstract/Free Full Text]
45. Brauker, J., Trautman, M., and Bernfield, M. (1991) Dev. Biol. 147, 285-292[CrossRef][Medline] [Order article via Infotrieve]
46. Ray, P., Tang, W., Wang, P., Homer, R., Kuhn, C., III, Flavell, R. A., and Elias, J. A. (1997) J. Clin. Invest. 100, 2501-2511[Medline] [Order article via Infotrieve]

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