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J Biol Chem, Vol. 275, Issue 16, 11858-11864, April 21, 2000
From the Children's Hospital Medical Center, Division of Pulmonary
Biology, Cincinnati, Ohio 45229-3039
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.
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.
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 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.
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).
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).
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.
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).
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-3 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.
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-3
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.
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.
*
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.
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.
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.
Conditional Expression of Fibroblast Growth Factor-7 in the
Developing and Mature Lung*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin are as
follows: -actin primer 1, 5'-GTG GGC CGC TCT AGG CAC CAA-3';
-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 -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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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[in a new window]
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.
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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).
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[in a new window]
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).
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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[in a new window]
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.
View larger version (57K):
[in a new window]
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 -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 -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
, 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).
ACKNOWLEDGEMENTS
FOOTNOTES
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.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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 X. Ye, J. Ding, X. Zhou, G. Chen, and S. F. Liu Divergent roles of endothelial NF-{kappa}B in multiple organ injury and bacterial clearance in mouse models of sepsis J. Exp. Med., June 9, 2008; 205(6): 1303 - 1315. [Abstract] [Full Text] [PDF]
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 W. D. Hardie, C. Davidson, M. Ikegami, G. D. Leikauf, T. D. Le Cras, A. Prestridge, J. A. Whitsett, and T. R. Korfhagen EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-{alpha}-induced pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1217 - L1225. [Abstract] [Full Text] [PDF]
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T. Chikama, C.-Y. Liu, J. T.A. Meij, Y. Hayashi, I-J. Wang, L. Yang, T. Nishida, and W. W.Y. Kao Excess FGF-7 in Corneal Epithelium Causes Corneal Intraepithelial Neoplasia in Young Mice and Epithelium Hyperplasia in Adult Mice Am. J. Pathol., March 1, 2008; 172(3): 638 - 649. [Abstract] [Full Text] [PDF]
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 A. C. White, K. J. Lavine, and D. M. Ornitz FGF9 and SHH regulate mesenchymal Vegfa expression and development of the pulmonary capillary network Development, October 15, 2007; 134(20): 3743 - 3752. [Abstract] [Full Text] [PDF]
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W. D. Hardie, T. R. Korfhagen, M. A. Sartor, A. Prestridge, M. Medvedovic, T. D. Le Cras, M. Ikegami, S. C. Wesselkamper, C. Davidson, M. Dietsch, et al. Genomic Profile of Matrix and Vasculature Remodeling in TGF-{alpha} Induced Pulmonary Fibrosis Am. J. Respir. Cell Mol. Biol., September 1, 2007; 37(3): 309 - 321. [Abstract] [Full Text] [PDF]
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 Y. Weng, C. Fang, R. J. Turesky, M. Behr, L. S. Kaminsky, and X. Ding Determination of the Role of Target Tissue Metabolism in Lung Carcinogenesis Using Conditional Cytochrome P450 Reductase-Null Mice Cancer Res., August 15, 2007; 67(16): 7825 - 7832. [Abstract] [Full Text] [PDF]
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E. L. Kramer, G. H. Deutsch, M. A. Sartor, W. D. Hardie, M. Ikegami, T. R. Korfhagen, and T. D. Le Cras Perinatal increases in TGF-{alpha} disrupt the saccular phase of lung morphogenesis and cause remodeling: microarray analysis Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L314 - L327. [Abstract] [Full Text] [PDF]
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Y. Zhang, G. Huang, L. P. Shornick, W. T. Roswit, J. M. Shipley, S. L. Brody, and M. J. Holtzman A Transgenic FOXJ1-Cre System for Gene Inactivation in Ciliated Epithelial Cells Am. J. Respir. Cell Mol. Biol., May 1, 2007; 36(5): 515 - 519. [Abstract] [Full Text] [PDF]
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K. Bry, J. A. Whitsett, and U. Lappalainen IL-1beta Disrupts Postnatal Lung Morphogenesis in the Mouse Am. J. Respir. Cell Mol. Biol., January 1, 2007; 36(1): 32 - 42. [Abstract] [Full Text] [PDF]
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 J. Xu, J. Tian, S. M. Grumelli, K. J. Haley, and S. D. Shapiro Stage-specific Effects of cAMP Signaling during Distal Lung Epithelial Development J. Biol. Chem., December 15, 2006; 281(50): 38894 - 38904. [Abstract] [Full Text] [PDF]
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H. Akei, J. A. Whitsett, M. Buroker, T. Ninomiya, H. Tatsumi, T. E. Weaver, and M. Ikegami Surface tension influences cell shape and phagocytosis in alveolar macrophages Am J Physiol Lung Cell Mol Physiol, October 1, 2006; 291(4): L572 - L579. [Abstract] [Full Text] [PDF]
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 K. K. Kim, M. C. Kugler, P. J. Wolters, L. Robillard, M. G. Galvez, A. N. Brumwell, D. Sheppard, and H. A. Chapman Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix PNAS, August 29, 2006; 103(35): 13180 - 13185. [Abstract] [Full Text] [PDF]
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 A. Dutt and K.-K. Wong Mouse models of lung cancer. Clin. Cancer Res., July 15, 2006; 12(14): 4396s - 4402s. [Abstract] [Full Text] [PDF]
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 P. W. Finch and J. S. Rubin Keratinocyte growth factor expression and activity in cancer: implications for use in patients with solid tumors. J Natl Cancer Inst, June 21, 2006; 98(12): 812 - 824. [Abstract] [Full Text] [PDF]
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 C. R. Hayworth, S. E. Moody, L. A. Chodosh, P. Krieg, M. Rimer, and W. J. Thompson Induction of neuregulin signaling in mouse schwann cells in vivo mimics responses to denervation. J. Neurosci., June 21, 2006; 26(25): 6873 - 6884. [Abstract] [Full Text] [PDF]
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 K. Politi, M. F. Zakowski, P.-D. Fan, E. A. Schonfeld, W. Pao, and H. E. Varmus Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors Genes & Dev., June 1, 2006; 20(11): 1496 - 1510. [Abstract] [Full Text] [PDF]
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H. Ji, X. Zhao, Y. Yuza, T. Shimamura, D. Li, A. Protopopov, B. L. Jung, K. McNamara, H. Xia, K. A. Glatt, et al. Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors PNAS, May 16, 2006; 103(20): 7817 - 7822. [Abstract] [Full Text] [PDF]
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T. H. Sisson, J. M. Hansen, M. Shah, K. E. Hanson, M. Du, T. Ling, R. H. Simon, and P. J. Christensen Expression of the Reverse Tetracycline-Transactivator Gene Causes Emphysema-Like Changes in Mice Am. J. Respir. Cell Mol. Biol., May 1, 2006; 34(5): 552 - 560. [Abstract] [Full Text] [PDF]
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A. C. White, J. Xu, Y. Yin, C. Smith, G. Schmid, and D. M. Ornitz FGF9 and SHH signaling coordinate lung growth and development through regulation of distinct mesenchymal domains Development, April 15, 2006; 133(8): 1507 - 1517. [Abstract] [Full Text] [PDF]
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 D. S. Basseres, E. Levantini, H. Ji, S. Monti, S. Elf, T. Dayaram, M. Fenyus, O. Kocher, T. Golub, K.-k. Wong, et al. Respiratory Failure Due to Differentiation Arrest and Expansion of Alveolar Cells following Lung-Specific Loss of the Transcription Factor C/EBP{alpha} in Mice Mol. Cell. Biol., February 1, 2006; 26(3): 1109 - 1123. [Abstract] [Full Text] [PDF]
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H. S. Floyd, C. L. Farnsworth, N. D. Kock, M. C. Mizesko, J. L. Little, S. T. Dance, J. Everitt, J. Tichelaar, J. A. Whitsett, and M. S. Miller Conditional expression of the mutant Ki-rasG12C allele results in formation of benign lung adenomas: development of a novel mouse lung tumor model Carcinogenesis, December 1, 2005; 26(12): 2196 - 2206. [Abstract] [Full Text] [PDF]
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M. Ikegami, J. A. Whitsett, P. C. Martis, and T. E. Weaver Reversibility of lung inflammation caused by SP-B deficiency Am J Physiol Lung Cell Mol Physiol, December 1, 2005; 289(6): L962 - L970. [Abstract] [Full Text] [PDF]
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M. L. Mucenski, J. M. Nation, A. R. Thitoff, V. Besnard, Y. Xu, S. E. Wert, N. Harada, M. M. Taketo, M. T. Stahlman, and J. A. Whitsett {beta}-Catenin regulates differentiation of respiratory epithelial cells in vivo Am J Physiol Lung Cell Mol Physiol, December 1, 2005; 289(6): L971 - L979. [Abstract] [Full Text] [PDF]
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A.-K. T. Perl, S. E. Wert, D. E. Loudy, Z. Shan, P. A. Blair, and J. A. Whitsett Conditional Recombination Reveals Distinct Subsets of Epithelial Cells in Trachea, Bronchi, and Alveoli Am. J. Respir. Cell Mol. Biol., November 1, 2005; 33(5): 455 - 462. [Abstract] [Full Text] [PDF]
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M. Ikegami, T. D. Le Cras, W. D. Hardie, M. T. Stahlman, J. A. Whitsett, and T. R. Korfhagen TGF-{alpha} perturbs surfactant homeostasis in vivo Am J Physiol Lung Cell Mol Physiol, July 1, 2005; 289(1): L34 - L43. [Abstract] [Full Text] [PDF]
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H.-M. I. Yu, B. Liu, S.-Y. Chiu, F. Costantini, and W. Hsu Development of a unique system for spatiotemporal and lineage-specific gene expression in mice PNAS, June 14, 2005; 102(24): 8615 - 8620. [Abstract] [Full Text] [PDF]
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I.-M. Kim, S. Ramakrishna, G. A. Gusarova, H. M. Yoder, R. H. Costa, and V. V. Kalinichenko The Forkhead Box M1 Transcription Factor Is Essential for Embryonic Development of Pulmonary Vasculature J. Biol. Chem., June 10, 2005; 280(23): 22278 - 22286. [Abstract] [Full Text] [PDF]
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 T.-i. Chikama, Y. Hayashi, C.-Y. Liu, N. Terai, K. Terai, C. W.-C. Kao, L. Wang, M. Hayashi, T. Nishida, P. Sanford, et al. Characterization of Tetracycline-Inducible Bitransgenic Krt12rtTA/+/tet-O-LacZ Mice Invest. Ophthalmol. Vis. Sci., June 1, 2005; 46(6): 1966 - 1972. [Abstract] [Full Text] [PDF]
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X. Lian, Y. Qin, S. A. Hossain, L. Yang, A. White, H. Xu, J. M. Shipley, T. Li, R. M. Senior, H. Du, et al. Overexpression of Stat3C in Pulmonary Epithelium Protects against Hyperoxic Lung Injury J. Immunol., June 1, 2005; 174(11): 7250 - 7256. [Abstract] [Full Text] [PDF]
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L. L. Nesslein, K. R. Melton, M. Ikegami, C.-L. Na, S. E. Wert, W. R. Rice, J. A. Whitsett, and T. E. Weaver Partial SP-B deficiency perturbs lung function and causes air space abnormalities Am J Physiol Lung Cell Mol Physiol, June 1, 2005; 288(6): L1154 - L1161. [Abstract] [Full Text] [PDF]
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S. M. Pope, P. C. Fulkerson, C. Blanchard, H. S. Akei, N. M. Nikolaidis, N. Zimmermann, J. D. Molkentin, and M. E. Rothenberg Identification of a Cooperative Mechanism Involving Interleukin-13 and Eotaxin-2 in Experimental Allergic Lung Inflammation J. Biol. Chem., April 8, 2005; 280(14): 13952 - 13961. [Abstract] [Full Text] [PDF]
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U. Lappalainen, J. A. Whitsett, S. E. Wert, J. W. Tichelaar, and K. Bry Interleukin-1{beta} Causes Pulmonary Inflammation, Emphysema, and Airway Remodeling in the Adult Murine Lung Am. J. Respir. Cell Mol. Biol., April 1, 2005; 32(4): 311 - 318. [Abstract] [Full Text] [PDF]
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R. Meuwissen and A. Berns Mouse models for human lung cancer Genes & Dev., March 15, 2005; 19(6): 643 - 664. [Abstract] [Full Text] [PDF]
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M. Ikegami, C.-L. Na, T. R. Korfhagen, and J. A. Whitsett Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells Am J Physiol Lung Cell Mol Physiol, March 1, 2005; 288(3): L552 - L561. [Abstract] [Full Text] [PDF]
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D. Spight, B. Zhao, M. Haas, S. Wert, A. Denenberg, and T. P. Shanley Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L251 - L265. [Abstract] [Full Text] [PDF]
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T. D. Le Cras, W. D. Hardie, G. H. Deutsch, K. H. Albertine, M. Ikegami, J. A. Whitsett, and T. R. Korfhagen Transient induction of TGF-{alpha} disrupts lung morphogenesis, causing pulmonary disease in adulthood Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L718 - L729. [Abstract] [Full Text] [PDF]
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 D. Gou, T. Narasaraju, N. R. Chintagari, N. Jin, P. Wang, and L. Liu Gene silencing in alveolar type II cells using cell-specific promoter in vitro and in vivo Nucleic Acids Res., September 27, 2004; 32(17): e134 - e134. [Abstract] [Full Text] [PDF]
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N. E. King, N. Zimmermann, S. M. Pope, P. C. Fulkerson, N. M. Nikolaidis, A. Mishra, D. P. Witte, and M. E. Rothenberg Expression and Regulation of a Disintegrin and Metalloproteinase (ADAM) 8 in Experimental Asthma Am. J. Respir. Cell Mol. Biol., September 1, 2004; 31(3): 257 - 265. [Abstract] [Full Text] [PDF]
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K. A. Wikenheiser-Brokamp Rb family proteins differentially regulate distinct cell lineages during epithelial development Development, September 1, 2004; 131(17): 4299 - 4310. [Abstract] [Full Text] [PDF]
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P. R. Reynolds, M. L. Mucenski, T. D. Le Cras, W. C. Nichols, and J. A. Whitsett Midkine Is Regulated by Hypoxia and Causes Pulmonary Vascular Remodeling J. Biol. Chem., August 27, 2004; 279(35): 37124 - 37132. [Abstract] [Full Text] [PDF]
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Mechanisms and Limits of Induced Postnatal Lung Growth Am. J. Respir. Crit. Care Med., August 1, 2004; 170(3): 319 - 343. [Full Text] [PDF]
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T. D. Le Cras, R. E. Spitzmiller, K. H. Albertine, J. M. Greenberg, J. A. Whitsett, and A. L. Akeson VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L134 - L142. [Abstract] [Full Text] [PDF]
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 L. Yang, X. Lian, A. Cowen, H. Xu, H. Du, and C. Yan Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-{alpha} in Regulation of the Surfactant Protein B Gene in the Lung Mol. Endocrinol., June 1, 2004; 18(6): 1520 - 1532. [Abstract] [Full Text] [PDF]
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A. Yu. Nikitin, A. Alcaraz, M. R. Anver, R. T. Bronson, R. D. Cardiff, D. Dixon, A. E. Fraire, E. W. Gabrielson, W. T. Gunning, D. C. Haines, et al. Classification of Proliferative Pulmonary Lesions of the Mouse: Recommendations of the Mouse Models of Human Cancers Consortium Cancer Res., April 1, 2004; 64(7): 2307 - 2316. [Abstract] [Full Text] [PDF]
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W. D. Hardie, T. D. Le Cras, K. Jiang, J. W. Tichelaar, M. Azhar, and T. R. Korfhagen Conditional expression of transforming growth factor-{alpha} in adult mouse lung causes pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L741 - L749. [Abstract] [Full Text] [PDF]
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V. V. Kalinichenko, G. A. Gusarova, I.-M. Kim, B. Shin, H. M. Yoder, J. Clark, A. M. Sapozhnikov, J. A. Whitsett, and R. H. Costa Foxf1 haploinsufficiency reduces Notch-2 signaling during mouse lung development Am J Physiol Lung Cell Mol Physiol, March 1, 2004; 286(3): L521 - L530. [Abstract] [Full Text] [PDF]
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I. Hokuto, A.-K. T. Perl, and J. A. Whitsett FGF signaling is required for pulmonary homeostasis following hyperoxia Am J Physiol Lung Cell Mol Physiol, March 1, 2004; 286(3): L580 - L587. [Abstract] [Full Text] [PDF]
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H. Wan, K. H. Kaestner, S.-L. Ang, M. Ikegami, F. D. Finkelman, M. T. Stahlman, P. C. Fulkerson, M. E. Rothenberg, and J. A. Whitsett Foxa2 regulates alveolarization and goblet cell hyperplasia Development, February 15, 2004; 131(4): 953 - 964. [Abstract] [Full Text] [PDF]
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C. J. Bachurski, G. H. Yang, T. A. Currier, R. M. Gronostajski, and D. Hong Nuclear Factor I/Thyroid Transcription Factor 1 Interactions Modulate Surfactant Protein C Transcription Mol. Cell. Biol., December 15, 2003; 23(24): 9014 - 9024. [Abstract] [Full Text] [PDF]
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M. L. Mucenski, S. E. Wert, J. M. Nation, D. E. Loudy, J. Huelsken, W. Birchmeier, E. E. Morrisey, and J. A. Whitsett {beta}-Catenin Is Required for Specification of Proximal/Distal Cell Fate during Lung Morphogenesis J. Biol. Chem., October 10, 2003; 278(41): 40231 - 40238. [Abstract] [Full Text] [PDF]
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K. R. Melton, L. L. Nesslein, M. Ikegami, J. W. Tichelaar, J. C. Clark, J. A. Whitsett, and T. E. Weaver SP-B deficiency causes respiratory failure in adult mice Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L543 - L549. [Abstract] [Full Text] [PDF]
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J. M. Roper, R. J. Staversky, J. N. Finkelstein, P. C. Keng, and M. A. O'Reilly Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L691 - L700. [Abstract] [Full Text] [PDF]
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 T. Shigehara, C. Zaragoza, C. Kitiyakara, H. Takahashi, H. Lu, M. Moeller, L. B. Holzman, and J. B. Kopp Inducible Podocyte-Specific Gene Expression in Transgenic Mice J. Am. Soc. Nephrol., August 1, 2003; 14(8): 1998 - 2003. [Abstract] [Full Text] [PDF]
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 L. Yang, A. Naltner, and C. Yan Overexpression of Dominant Negative Retinoic Acid Receptor {alpha} Causes Alveolar Abnormality in Transgenic Neonatal Lungs Endocrinology, July 1, 2003; 144(7): 3004 - 3011. [Abstract] [Full Text] [PDF]
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P. Ray, Y. Devaux, D. B. Stolz, M. Yarlagadda, S. C. Watkins, Y. Lu, L. Chen, X.-f. Yang, and A. Ray Inducible expression of keratinocyte growth factor (KGF) in mice inhibits lung epithelial cell death induced by hyperoxia PNAS, May 13, 2003; 100(10): 6098 - 6103. [Abstract] [Full Text] [PDF]
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 T. A. Tucker, K. Varga, Z. Bebok, A. Zsembery, N. A. McCarty, J. F. Collawn, E. M. Schwiebert, and L. M. Schwiebert Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids Am J Physiol Cell Physiol, March 1, 2003; 284(3): C791 - C804. [Abstract] [Full Text] [PDF]
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I. Hokuto, A.-K. T. Perl, and J. A. Whitsett Prenatal, but Not Postnatal, Inhibition of Fibroblast Growth Factor Receptor Signaling Causes Emphysema J. Biol. Chem., January 3, 2003; 278(1): 415 - 421. [Abstract] [Full Text] [PDF]
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 H. Fehrenbach, A. Fehrenbach, T. Pan, M. Kasper, and R.J. Mason Keratinocyte growth factor-induced proliferation of rat airway epithelium is restricted to Clara cells in vivo Eur. Respir. J., November 1, 2002; 20(5): 1185 - 1197. [Abstract] [Full Text] [PDF]
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T. H. Sisson, K. E. Hanson, N. Subbotina, A. Patwardhan, N. Hattori, and R. H. Simon Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L1023 - L1032. [Abstract] [Full Text] [PDF]
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L. Zhang, M. Ikegami, C. R. Dey, T. R. Korfhagen, and J. A. Whitsett Reversibility of Pulmonary Abnormalities by Conditional Replacement of Surfactant Protein D (SP-D) in Vivo J. Biol. Chem., October 4, 2002; 277(41): 38709 - 38713. [Abstract] [Full Text] [PDF]
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F. Demayo, P. Minoo, C. G. Plopper, L. Schuger, J. Shannon, and J. S. Torday Mesenchymal-epithelial interactions in lung development and repair: are modeling and remodeling the same process? Am J Physiol Lung Cell Mol Physiol, September 1, 2002; 283(3): L510 - L517. [Abstract] [Full Text] [PDF]
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A.-K. T. Perl, S. E. Wert, A. Nagy, C. G. Lobe, and J. A. Whitsett Early restriction of peripheral and proximal cell lineages during formation of the lung PNAS, August 6, 2002; 99(16): 10482 - 10487. [Abstract] [Full Text] [PDF]
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C. Liu, E. E. Morrisey, and J. A. Whitsett GATA-6 is required for maturation of the lung in late gestation Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L468 - L475. [Abstract] [Full Text] [PDF]
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J. A. Whitsett, J. C. Clark, L. Picard, J. W. Tichelaar, S. E. Wert, N. Itoh, A.-K. T. Perl, and M. T. Stahlman Fibroblast Growth Factor 18 Influences Proximal Programming during Lung Morphogenesis J. Biol. Chem., June 14, 2002; 277(25): 22743 - 22749. [Abstract] [Full Text] [PDF]
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L. B. Ware and M. A. Matthay Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair Am J Physiol Lung Cell Mol Physiol, May 1, 2002; 282(5): L924 - L940. [Abstract] [Full Text] [PDF]
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B. Zheng, Z. Zhang, C. M. Black, B. de Crombrugghe, and C. P. Denton Ligand-Dependent Genetic Recombination in Fibroblasts : A Potentially Powerful Technique for Investigating Gene Function in Fibrosis Am. J. Pathol., May 1, 2002; 160(5): 1609 - 1617. [Abstract] [Full Text] [PDF]
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 N. Sato, K. Matsuda, C. Sakuma, D. N. Foster, R. W. Oppenheim, and H. Yaginuma Regulated Gene Expression in the Chicken Embryo by Using Replication-Competent Retroviral Vectors J. Virol., February 15, 2002; 76(4): 1980 - 1985. [Abstract] [Full Text] [PDF]
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K. S. Harrod and R. J. Jaramillo Pseudomonas aeruginosa and Tumor Necrosis Factor-alpha Attenuate Clara Cell Secretory Protein Promoter Function Am. J. Respir. Cell Mol. Biol., February 1, 2002; 26(2): 216 - 223. [Abstract] [Full Text] [PDF]
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H. Yang, M. M. Lu, L. Zhang, J. A. Whitsett, and E. E. Morrisey GATA6 regulates differentiation of distal lung epithelium Development, January 5, 2002; 129(9): 2233 - 2246. [Abstract] [Full Text] [PDF]
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G. H. Fisher, S. L. Wellen, D. Klimstra, J. M. Lenczowski, J. W. Tichelaar, M. J. Lizak, J. A. Whitsett, A. Koretsky, and H. E. Varmus Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes Genes & Dev., December 15, 2001; 15(24): 3249 - 3262. [Abstract] [Full Text] [PDF]
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 J. A. Whitsett, S. W. Glasser, J. W. Tichelaar, A.-K. T. Perl, J. C. Clark, and S. E. Wert Transgenic Models for Study of Lung Morphogenesis and Repair : Parker B. Francis Lecture Chest, July 1, 2001; 120(2007): 27S - 30S. [Full Text] [PDF]
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C. A. Cronin, W. Gluba, and H. Scrable The lac operator-repressor system is functional in the mouse Genes & Dev., June 15, 2001; 15(12): 1506 - 1517. [Abstract] [Full Text] [PDF]
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R. H. Costa, V. V. Kalinichenko, and L. Lim Transcription factors in mouse lung development and function Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L823 - L838. [Abstract] [Full Text] [PDF]
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B. Zhao, S. S. Chua, M. M. Burcin, S. D. Reynolds, B. R. Stripp, R. A. Edwards, M. J. Finegold, S. Y. Tsai, and F. J. DeMayo Phenotypic consequences of lung-specific inducible expression of FGF-3 PNAS, April 25, 2001; (2001) 101116598. [Abstract] [Full Text]
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J. C. Clark, J. W. Tichelaar, S. E. Wert, N. Itoh, A.-K. T. Perl, M. T. Stahlman, and J. A. Whitsett FGF-10 disrupts lung morphogenesis and causes pulmonary adenomas in vivo Am J Physiol Lung Cell Mol Physiol, April 1, 2001; 280(4): L705 - L715. [Abstract] [Full Text] [PDF]
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Z. Zhu, B. Ma, R. J. Homer, T. Zheng, and J. A. Elias Use of the Tetracycline-controlled Transcriptional Silencer (tTS) to Eliminate Transgene Leak in Inducible Overexpression Transgenic Mice J. Biol. Chem., June 29, 2001; 276(27): 25222 - 25229. [Abstract] [Full Text] [PDF]
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B. Zhao, S. S. Chua, M. M. Burcin, S. D. Reynolds, B. R. Stripp, R. A. Edwards, M. J. Finegold, S. Y. Tsai, and F. J. DeMayo Phenotypic consequences of lung-specific inducible expression of FGF-3 PNAS, May 8, 2001; 98(10): 5898 - 5903. [Abstract] [Full Text] [PDF]
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