FGF-10 disrupts lung morphogenesis and causes pulmonary
adenomas in vivo
Jean C.
Clark1,
Jay W.
Tichelaar1,
Susan E.
Wert1,
Nobuyuki
Itoh2,
Anne-Karina T.
Perl1,
Mildred T.
Stahlman3, and
Jeffrey A.
Whitsett1
1 Division of Pulmonary Biology, Children's Hospital
Medical Center, Cincinnati, Ohio 45229-3039; 2 Department of
Genetic Biochemistry, Kyoto University Graduate School of
Pharmaceutical Sciences, Kyoto 606-8501, Japan; and 3 Department
of Pediatrics, Vanderbilt University, Nashville, Tennessee
37232-2370
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ABSTRACT |
Transgenic mice in which fibroblast growth factor
(FGF)-10 was expressed in the lungs of fetal and postnatal mice were
generated with a doxycycline-inducible system controlled by surfactant
protein (SP) C or Clara cell secretory protein (CCSP) promoter
elements. Expression of FGF-10 mRNA in the fetal lung caused
adenomatous malformations, perturbed branching morphogenesis, and
caused respiratory failure at birth. When expressed after birth, FGF-10
caused multifocal pulmonary tumors. FGF-10-induced tumors were highly
differentiated papillary and lepidic pulmonary adenomas. Epithelial
cells lining the tumors stained intensely for thyroid transcription
factor (TTF)-1 and SP-C but not CCSP, indicating that FGF-10 enhanced differentiation of cells to a peripheral alveolar type II cell phenotype. Withdrawal from doxycycline caused rapid regression of the
tumors associated with rapid loss of the differentiation markers TTF-1,
SP-B, and proSP-C. FGF-10 disrupted lung morphogenesis and induced
multifocal pulmonary tumors in vivo and caused reversible type II cell
differentiation of the respiratory epithelium.
fibroblast growth factor; conditional expression
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INTRODUCTION |
THE LUNG BUD EVAGINATES
FROM the foregut endoderm and undergoes stereotypic dichotomous
branching as it invades the splanchnic mesenchyme. Proliferation and
branching of the lung buds require inductive signals provided by the
mesenchyme that are mediated in part by the binding of members of the
fibroblast growth factor (FGF) family of polypeptides to FGF receptors
(FGFR) on target cells (for review see Refs. 17,
24, and 38). Both in vivo and in vitro experiments
support the importance of FGF signaling in lung morphogenesis. FGF
polypeptides, including FGF-1, FGF-2, FGF-7, FGF-9, FGF-10, and FGF-18,
are expressed in the developing lung (1, 2, 9, 14, 23, 26,
33) as are FGF receptors FGFR1, FGFR2, FGFR3, and FGFR4
(13, 19, 27, 30, 31). The FGFR2-IIIb splice variant is
expressed at high concentrations in the epithelial cells of the lung
buds (1), likely binding FGF-7 and FGF-10 expressed by
mesenchymal cells. The primary role of FGF-10 in lung morphogenesis was
demonstrated by the finding that targeted disruption of the murine
FGF-10 gene blocked lung and limb morphogenesis in vivo (7, 25,
34). In the mouse, FGF-10 mRNA was detected in spatially
restricted sites at the tips of the lung buds, in close apposition to
the sites of expression of FGFR2 (2), known to bind both
FGF-7 and FGF-10 (15). Respiratory epithelial cells of the
fetal lung migrated toward FGF-10-coated beads (28), and
budding was induced by FGF-10 and FGF-7 in vitro (2).
Taken together, these studies support the concept that the precise
temporospatial expression of FGF-10 might mediate the sites and extent
of branching morphogenesis in the developing lung. The finding that FGF
signaling was required for lung formation in the mouse was further
supported by observations in Drosophila, wherein disruption
of the branchless gene (a FGF homolog) or the breathless gene (an FGFR homolog), inhibited migration
of cells of the tracheal system and blocked tracheal tube formation and branching (32, 37).
Various FGF polypeptides are expressed in the lung with distinct
temporospatial patterns of expression. For example, FGF-7 (also termed
keratinocyte growth factor) is expressed by fetal lung mesenchyme
(8, 23). Ectopic expression of FGF-7 in the developing
lung in vivo or application of FGF-7 to lung explants in vitro
disrupted branching morphogenesis and enhanced epithelial cell
proliferation, causing cystadenomatoid malformations (35, 44). Postnatally, intratracheal administration of FGF-7 caused diffuse alveolar and bronchiolar cell hyperplasia that resolved rapidly, demonstrating the sensitivity of the lung to increased FGF
signaling (41). Despite the marked proliferative effects of FGF-7 in vivo, morphogenesis of the fetal lung was not perturbed in
FGF-7 gene-inactivated mice, suggesting redundant activity of FGF
polypeptides or lack of a requirement for FGF-7 in lung formation
(12). FGF-1, FGF-2, FGF-9, FGF-10, and FGF-18 are also
expressed in the developing lung. The precise roles of each of these
polypeptides and whether they serve distinct or overlapping functions
in lung morphogenesis or repair are not known at present.
Most FGF polypeptides bind with overlapping specificity to FGFR. FGF-7
is unique in binding selectively to FGFR2-IIIb; FGF-10, however, binds
and activates both FGFR2-IIIb and FGFR1-IIIb in vitro
(22). Expression of an FGFR dominant negative receptor with the surfactant protein (SP) C promoter in vivo blocked branching morphogenesis of the lung and was associated with complete loss of the
distal subset of respiratory epithelial cells, demonstrating a critical
requirement for FGFR signaling in lung morphogenesis (29).
Likewise, expression of a soluble FGFR dominant negative mutant blocked
limb and lung development in vivo, findings identical to those in the
FGF-10-null mice, supporting the primary role of FGFR signaling and
FGF-10 in lung morphogenesis (6). Whereas FGFR3- and
FGFR4-null mice did not have abnormalities in lung formation,
double-null FGFR3/4 mice developed emphysema in the postnatal period,
demonstrating a more subtle effect of FGFR3 and FGFR4 on postnatal lung
growth (42). The ligands mediating FGFR3 and FGFR4
signaling have not been clarified. It therefore remains unclear how the
precise temporal, spatial, and stoichiometric expression of FGF
polypeptides mediate normal branching morphogenesis of the lung in vivo.
In the present study, conditional expression of FGF-10 was achieved
utilizing the reverse tetracycline transactivator (rtTA) (11) that is expressed in respiratory epithelial cells
under control of either the SP-C or Clara cell secretory protein (CCSP) promoters. Expression of FGF-10 in the fetal lung altered lung morphogenesis and caused focal pulmonary adenomas when induced in the
postnatal period.
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EXPERIMENTAL PROCEDURES |
Transgenic mice.
Permanent transgenic mouse lines bearing the SP-C-rtTA and CCSP-rtTA
transgenes were established on an FVB/N background after oocyte
injection of the plasmid constructs. The constructs consist of 3.7 kb
of the human SP-C promoter (10) or 2.3 kb of the rat CCSP
promoter (36) placed 5' of the rtTA gene construct. The mouse FGF-10 cDNA (39) was inserted between the
(teto)7CMV promoter and the 3'-untranslated region of
the bovine growth hormone gene (Fig.
1). The rtTA and (teto)7
constructs were kindly provided by Dr. Herman Bujard (Heidelberg)
(11). Offspring of all founders were screened by Southern
blot analysis; mice transmitting the "activator" rtTA transgene
were bred to establish permanent CCSP-rtTA and SP-C-rtTA mouse colonies
(40). Transgenic CCSP and SP-C-rtTA activator lines have
been stable for more than a year in the vivarium. The target
mice, (teto)7CMV-FGF-10, were generated by oocyte injection of the plasmid DNA into the FVB/N strain, and founders were
screened by PCR analysis. Heterozygous
(teto)7CMV-FGF-10 mice were viable and without observable
abnormalities. Two separate target lines bearing the
(teto)7CMV-FGF-10 transgene (lines A and
B) were chosen for breeding to the CCSP-rtTA and SP-C-rtTA
activator mice, transmitting the genes with typical Mendelian
inheritance patterns. All mice were maintained in a pathogen-free
vivarium. Doxycycline (0.5 mg/ml) was administered in drinking water
for the described time periods, the solution being changed three times
per week.

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Fig. 1.
Design of transgenes for conditional expression of mouse (m)
fibroblast growth factor (FGF)-10. Promoters utilized for expression of
reverse tetracycline transactivator (rtTA) in respiratory epithelial
cells consisted of 3.7 kb of the 5'-flanking region of human (h)
surfactant protein (SP) C gene or 2.3 kb of the 5'-flanking region of
the rat (r) Clara cell secretory protein (CCSP) gene. The 3' region of
the transgenes consisted of 3'-untranslated and polyadenylation
sequences from the SV40 large T region for SP-C-rtTA or human growth
hormone (hGH) for CCSP-rtTA. The target construct consisted of the
concatamerized (teto)7 and a minimal CMV promoter, the
murine FGF-10 cDNA and termination sequences from the bovine growth
hormone (bGH) gene. Activator mice were mated to either of 2 (teto)7CMV-FGF-10 target lines (lines A or
B) to generate double-transgenic mice expressing FGF-10 mRNA
in a doxycycline-inducible manner.
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RT-PCR.
Tissues were homogenized in TRIzol reagent (Life Technologies), and RNA
was isolated according to the manufacturer's specification. RNA was
treated with DNase before cDNA synthesis. RNA (5 µg) was reverse
transcribed and then analyzed by PCR for total FGF-10, exogenous
FGF-10, and
-actin mRNA. Transgene-specific primers for the mouse
FGF-10 were designed to the (teto)7CMV-FGF-10 transcript primer A in the CMV minimal promoter (5' to 3') GAC GCC ATC
CAC GCT GTT; [primer B in the FGF-10 cDNA (5' to 3') ATT
TGC CTG CCA TTG TGC TGC CAG], used for amplification, and compared
with those amplified for
-actin. Total FGF-10 was measured using
primers designed to amplify within the FGF-10 coding sequence.
Histology, immunohistochemistry, and electron microscopy.
To obtain fetal lung tissue, the fetuses were removed by hysterotomy
after lethal injection of pentobarbital sodium to the dam. The chest of
fetal animals was opened, and the tissue was fixed with 4%
paraformaldehyde at 4°C. Lungs from postnatal animals were inflation
fixed at 25 cmH2O pressure via a tracheal cannula with the
same fixative. Tissue was fixed overnight, washed in PBS, dehydrated
through a series of alcohols, and embedded in paraffin. Tissue sections
were stained for SP-B, proSP-B, thyroid transcription factor (TTF)-1,
proSP-C, CCSP, and 5-bromo-2'-deoxyuridine (BrdU) as previously
described (10). For electron microscopy, tissue was
postfixed in 1% osmium tetroxide and evaluated as previously described
(18).
In situ hybridization.
Expression of FGF-10 mRNA was assessed by in situ hybridization using
35S-labeled riboprobes as previously described for fetal
and adult lungs (40), the latter after inflation fixation
at 25 cmH2O pressure. Sense and antisense FGF-10 RNA probes
were generated in pGEM32. Tissue was hybridized overnight at 50°C.
Slides were coated with Kodak NTB-2 emulsion, exposed for 3-7
days, and developed with Kodak D19.
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RESULTS |
Generation of SP-C-rtTA, CCSP-rtTA, and
(teto)7- CMV-FGF-10 transgenic mice.
Transgenic CCSP-rtTA, SP-C-rtTA, and (teto)7CMV-FGF-10 mice
(heterozygous for each transgene) were viable and were produced in
ratios predicted by Mendelian inheritance. Characteristics of the rtTA
activator mice were recently described (40). In situ
hybridization analysis of the lungs from SP-C-rtTA activator mice
demonstrated that rtTA mRNA was selectively expressed in peripheral
respiratory epithelial cells in the lungs of fetal [postconception
(pc) day 15] and adult mice. In CCSP-rtTA mice, rtTA mRNA was detected in both tracheobronchial and type II cells (40). Two independent lines of
(teto)7CMV-FGF-10 target mice (lines A and
B) were generated.
Conditional expression of FGF-10 mRNA.
Transgene-specific FGF-10 mRNA was assessed by RT-PCR in lungs of young
adult mice with and without addition of 0.5 mg/ml doxycycline in the
drinking water. In adult double-transgenic CCSP-rtTA × (teto)7CMV-FGF-10 mice ("target" lines A and
B), FGF-10 mRNA was undetectable in the absence of
doxycycline but was markedly induced after oral doxycycline and
reversed by withdrawal from doxycycline (Fig.
2). In CCSP-rtTA × (teto)7CMV-FGF-10 mice (line B), FGF-10 mRNA was
detected in fetal and adult lung but transgene mRNA was detected only
in postnatal lung from mice generated from line A. FGF-10
mRNA was detected in SP-C-rtTA × (teto)7CMV-FGF-10 (line A) adult double-transgenic mice in the presence or
absence of doxycycline, the abundance of FGF-10 mRNA being increased by exposure to doxycycline; however, FGF-10 mRNA was not detected in fetal
lung (pc day 18) with or without doxycycline in
SP-C-rtTA × (teto)7CMV-FGF-10 line A
offspring (data not shown). In contrast, double-transgenic mice
generated by crossing SP-C-rtTA and (teto)7CMV-FGF-10 (line B) generally died at birth whether or not they were
treated with doxycycline, and abundant FGF-10 mRNA was detected in
lungs of these fetal mice at day 18 of gestation. Transgenic
FGF-10 mRNA was not detected in other major organs, including liver, kidney, brain, testes, muscle, and heart, typical of the specificity of
the CCSP and SP-C promoter elements, which are generally active only in
respiratory epithelial cells in the lung (10, 36) (data
not shown).

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Fig. 2.
Conditional expression of FGF-10 mRNA controlled by
doxycycline. Total lung RNA from adult CCSP-rtTA × (teto)7CMV-FGF-10 (line A) transgenic mice was
used to amplify mouse FGF-10. Primers were used to selectively amplify
the FGF-10 transgene (top), total mouse FGF-10 mRNA
(exogenous plus endogenous; middle), or -actin
(bottom). Lane 1, lung RNA from a
double-transgenic mouse in the absence of doxycycline. Lane
2, lung RNA from a single-transgenic (teto)7FGF-10
mouse on doxycycline for 4 wk. Lanes 3-5, lung RNA from
double-transgenic mice on doxycycline for 4 wk (n = 3).
Lanes 6-8, lung RNA from double-transgenic mice treated
with doxycycline for 4 wk and then removed from doxycycline for 4 wk
(n = 3).
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Effects of FGF-10 on the fetal lung.
Histology of lungs from double-transgenic SP-C or CCSP-rtTA × (teto)7-CMV-FGF-10 offspring (line B) was
assessed in fetal mice treated with doxycycline (Fig.
3). At pc day 17, histological abnormalities observed in the FGF-10-expressing mice were similar with
both SP-C- and CCSP-driven transgenes, consisting of marked adenomatoid
hyperplasia with filling of peripheral and small conducting airways
with hyperplastic epithelial cells. Whereas similar abnormalities were
observed in all FGF-10-expressing fetal mice, variability in the sites
and extent of hyperplasia was observed even in the same litter (Fig.
3), suggesting that the timing, extent, or levels of transgene
expression may vary and influence phenotype. In general, the fetal
lungs were highly cellular, consisting of dense lung parenchyma with
characteristics of focal or diffuse pulmonary adenomas. Occasionally,
cystic changes were observed in the lung parenchyma at pc day
17. Generally, epithelial cells lining the respiratory tubules of
FGF-10-expressing mice stained intensely for proSP-C, TTF-1, and SP-B,
consistent with the characteristics of type II epithelial cells (data
not shown). Multiple adenomatous polyps were occasionally observed in
the conducting airways of double-transgenic CCSP-rtTA pups on
doxycycline at pc day 17 (Fig. 3B), and some of
these cells stained for CCSP (data not shown). In contrast to
CCSP-rtTA × (teto)7CMV-FGF-10 (line B)
fetal pups, CCSP-rtTA × (teto)7CMV-FGF-10 (line
A) pups had normal lung histology following doxycycline treatment;
however, in situ hybridization analysis revealed a lack of mRNA
expression in this line in the fetal, but not in the adult, mouse
lung.

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Fig. 3.
FGF-10 caused malformations in
the fetal lung. Lungs from (teto)7CMV-FGF-10 (line
B) transgenic pups or double-transgenic CCSP-rtTA × (teto)7CMV-FGF-10 (line B) pups were assessed at
17 days postconception (pc) after being stained with hematoxylin and
eosin. The dam was continuously treated with doxycycline
postconception. Lung histology of single-transgenic mice shown in
A was not different from wild type. In distinct
double-transgenic mice (B-D), marked abnormalities in lung
structure, consisting of adenomatous hyperplasia, loss of peripheral
airspaces, disruption of branching morphogenesis, and conducting airway
adenomas (B) were observed in FGF-10-expressing mice.
Original magnification, ×40. Figures are representative of 4 mice.
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In situ hybridization demonstrated widespread expression of FGF-10 mRNA
in respiratory epithelial cell in conducting airway and alveolar
regions in fetal SP-C and CCSP-rtTA × (teto)7CMV-FGF-10 exposed to doxycycline (Fig.
4). FGF-10 mRNA was detected in fetal lung from CCSP-rtTA × (teto)7CMV-FGF-10
(line B) only in the presence of doxycycline but was
detected in SP-C-rtTA × (teto)7CMV-FGF-10 (line
B) in the presence and absence of doxycycline. Endogenous mouse
FGF-10 mRNA was not detected in control lung tissue under these same
hybridization and autoradiographic exposures, but mouse FGF-10 mRNA was
detected by RT-PCR in control lung (Fig. 4). Histological abnormalities
and FGF-10 mRNA levels in SP-C-rtTA × (teto)7CMV-FGF-10 (line B) lung were increased
by administration of doxycycline to the dam (data not shown).
SP-C-rtTA × (teto)7CMV-FGF-10 (line B)
mice generally did not survive postnatally, likely because of severe
pulmonary malformations. In contrast, FGF-10 mRNA was not detected in
the fetal lungs (pc day 17) of double-transgenic CCSP-rtTA × (teto)7CMV-FGF-10 (line A),
explaining the normal lung histology seen in these animals (data not
shown). In general, histological abnormalities seen in the
double-transgenic mice correlated with the level of expression of the
transgene.

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Fig. 4.
Localization of FGF-10 mRNA in fetal lung. FGF-10 mRNA
was detected in lungs of double-transgenic mice on day 17 pc
by in situ hybridization with a mouse FGF-10 mRNA probe. Dams were
maintained continuously on doxycycline postconception. FGF-10 mRNA was
detected in respiratory epithelial cells in lungs of double-transgenic
mice from (teto)7CMV-FGF-10 (line B) offspring,
using the SP-C-rtTA activator (A) or CCSP-rtTA activator
(B). Slides were exposed for 3 days. No signal was detected
when a sense riboprobe was used (C). Original magnification,
×40.
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FGF-10 caused multifocal pulmonary tumors postnatally.
In the absence of doxycycline, lung size and histology of
double-transgenic mice from CCSP-rtTA × (teto)7CMV-FGF-10 (lines A and B)
were not different from the nontransgenic controls. In contrast, when
weanling double-transgenic mice from either line A or
B were treated with doxycycline in the drinking water for 2-4 wk, all double-transgenic mice developed multifocal,
adenomatous lung tumors (Fig. 5). FGF-10
mRNA was readily detected in the tumors by in situ hybridization (Fig.
6). Abnormalities were confined to the
lung, and the number and size of tumors were generally greater in
double-transgenic mice from line B, consistent with greater
signal intensity of the transgenic mRNA in line B (data not
shown). Tumors were readily visible by gross inspection, and histological classification was consistent with multifocal lepidic and
papillary tumors (Figs. 5-7). Tumor
formation was extensive and observed in all double-transgenic mice
treated with doxycycline but was never seen in wild-type,
single-transgenic mice or double-transgenic CCSP-rtTA × (teto)7FGF-10 mice in the absence of doxycycline. Pulmonary
tumors and adenomatous changes were seen in lungs of all
double-transgenic SP-C-rtTA × (teto)7CMV-FGF-10
(line A) mice in the presence and absence of doxycycline,
number, and size of the tumors being increased by doxycycline
treatment. In these mice, tumors were generally observed in the lung
periphery and were not observed in larger conducting airways. The size
of the lungs increased, and dense, adenomatous tumors became extensive in SP-C-rtTA × (teto)7CMV-FGF-10 (line B)
after doxycycline administration (Fig. 5) and often caused respiratory
distress. Increased numbers of mononuclear cells were consistently
observed in the lung parenchyma surrounding the tumors in all
FGF-10-expressing mice. SP-C-rtTA × (teto)7CMV-FGF-10
(line B) mice rarely survived postnatally, consistent with
both the leaky expression of FGF-10 in SP-C-activated mice and the
increased severity of lung malformations in
(teto)7CMV-FGF-10 (line B) compared with
line A.

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Fig. 5.
FGF-10 caused tumors in the postnatal lung. Lungs were
inflation fixed from CCSP-rtTA × (teto)7CMV-FGF-10 (line A) mice
(top) or CCSP-rtTA × (teto)7CMV-FGF-10
(line B) mice (bottom), and the left lobe was
photographed. Line A mice were treated with doxycycline for
4 wk (+Dox) or for 4 wk followed by 4 wk without doxycycline
(+Dox/ Dox). Line B mice were treated for 12 days (+Dox) or
for 12 days followed by 6 days without doxycycline (+Dox/ Dox). Tumors
largely resolved after removal of doxycycline, demonstrating the
reversibility of the tumors induced by FGF-10. Figures are
representative of 3 separate experiments with each
line.
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Fig. 6.
In
situ hybridization of FGF-10 mRNA in adult lung. Tumors were induced by
placing weanling double-transgenic CCSP-rtTA × (teto)7CMV-FGF-10 (line A) on doxycycline for 4 wk. Lung tissue was hybridized with antisense (A) or sense
(B) FGF-10 RNA probes, demonstrating hybridization in
epithelial cells in the tumors. Original magnification, ×40.
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Fig. 7.
Immunohistochemistry of FGF-10-induced tumors. Tumors were induced
by treating adult double-transgenic CCSP-rtTA × (teto)7CMV-FGF-10 (line A) mice with doxycycline
for 4 wk. Tissue sections were stained for proSP-C, proSP-B, thyroid
transcription factor (TTF)-1, or CCSP. Tumors stained for type II
epithelial cell markers, proSP-C, and proSP-B but did not stain for
CCSP. Findings are representative of at least 3 separate mice with each
antibody. Original magnification, ×40.
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Respiratory epithelial markers.
Whereas much of the lung parenchyma was relatively unaffected,
focal adenomas were readily observed throughout the lungs of mice
expressing FGF-10 in the postnatal period. Epithelial cells in tumors
from adult double-transgenic CCSP- and SP-C-rtTA-activated mice stained
intensely for TTF-1, proSP-C, and proSP-B (Fig. 7). Generally,
epithelial cells in the relatively normal lung parenchyma surrounding
the lesions stained less intensely for each of these markers, but the
intensity of staining for TTF-1, proSP-B, and proSP-C was increased in
type II cells in the FGF-10-expressing mice compared with that in
wild-type mice. Tumor cells were stained by both proSP-B and mature
SP-B antisera (latter not shown), indicating that the cells processed
proSP-B to the active SP-B peptide, a function restricted to type II
epithelial cells in the normal lung (20). Consistent with
this observation, the intensity of staining for proSP-C, a type II
cell-specific marker, was increased in the tumors from both CCSP- and
SP-C-driven double-transgenic mice while staining for endogenous CCSP,
a marker of nonciliated bronchiolar cells, was not generally observed
in the tumors from postnatal animals. BrdU labeling was selectively
increased in the tumors from the double-transgenic mice (Fig.
8), but focal increased BrdU uptake was
occasionally observed in less involved regions of the lung parenchyma.

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Fig. 8.
5-Bromo-2'-deoxyuridine (BrdU)
staining in FGF-10-induced tumor. BrdU uptake was assessed in a
double-transgenic CCSP-rtTA × (teto)7CMV-FGF-10
(line A) adult mouse treated with doxycycline for 4 wk. The
animal was killed 2 h after BrdU injection and uptake was assessed
by immunohistochemistry. Labeling was consistently increased in the
tumors but was occasionally observed in relatively unaffected lung
parenchyma (data not shown). Findings are representative of at least 3 mice. Original magnification, ×200.
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Ultrastructure of FGF-10-induced tumors.
Ultrastructural features of the FGF-10-induced tumors were typical of
type II epithelial cell tumors (Fig. 9).
Tumor cells were predominantly cuboidal and contained numerous lamellar
bodies and extensive microvilli. Increased numbers of type II
epithelial cells were often located along alveolar septa in less
affected regions of the lung parenchyma, a finding generally not
observed in normal lung.

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Fig. 9.
Lung ultrastructure after FGF-10 expression. Electron microscopy
was performed on lung tissue from SP-C-rtTA × (teto)7CMV-FGF-10 (line A) double-transgenic
mouse as described in EXPERIMENTAL PROCEDURES.
A: large tumor cell with many lamellar bodies. B:
type II-like cells with secreted surfactant within the lumen.
C: aberrant location of a type II cell at the end of an
alveolar septum. D: type II cell tumor along an alveolar
septum.
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Reversal of doxycycline-induced tumor formation.
To assess the reversibility of the tumor formation, double
CCSP-rtTA × (teto)7CMV-FGF-10 transgenic mice were
placed on doxycycline for several weeks and withdrawn from doxycycline
for 1-4 wk (line A) or on doxycycline for 12 days and
withdrawn from doxycycline for 6 days (line B). Dramatic
tumor regression was observed in all animals after removal from
doxycycline (Figs. 5 and 10) in association with the loss of FGF-10 mRNA (Fig. 2). Residual focal abnormalities with increased numbers of mononuclear cells were observed
in the lung parenchyma, consistent with remodeling at the site of tumor
regression (Fig. 10), and fibrotic changes were not observed after
recovery.

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Fig. 10.
Reversal of FGF-10-induced tumor. Representative lung histology
from a double-transgenic CCSP-rtTA × (teto)7CMV-FGF-10 (line A) mouse treated for 4 wk with doxycycline and withdrawn from doxycycline for 4 wk. Regions of
remodeled lung parenchyma were readily observed in the lung periphery.
Figures are representative of at least 6 double-transgenic mice treated
and withdrawn from doxycycline. Previous sites of focal tumors
contained increased numbers of alveolar macrophages and abnormalities
of alveolar structure but with near-complete resolution of epithelial
cell hyperplasia. Original magnifications: ×40 (top); ×200
(bottom).
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Withdrawal from doxycycline caused rapid loss of cuboidal cell
morphology, decrease in intensity, and extent of expression of TTF-1,
proSP-C, and SP-B, consistent with loss of type II epithelial cell
characteristics (Fig. 11). After
removal from doxycycline, deoxynucleotidyltransferase dUTP nick end
labeling (TUNEL) assay and electron microscopy of the tumors did not
show evidence of large-scale apoptosis that could account for
the rapid resolution of the tumors (data not shown).

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Fig. 11.
TTF-1, proSP-C, and SP-B during
tumor regression. Representative immunostaining for TTF-1, proSP-C, and
SP-B is demonstrated during tumor regression in CCSP-rtTA × (teto)7CMV-FGF-10 mice (line A). Animals were
placed on doxycycline for 2 mo and withdrawn for 3-10 days.
Immunostaining for TTF-1, proSP-C, and SP-B decreased rapidly after
withdrawal from doxycycline. Initial magnification,
×100.
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DISCUSSION |
FGF-10 mRNA was selectively and conditionally expressed in
respiratory epithelial cells of the lungs of fetal and postnatal mice
under control of doxycycline. Pulmonary morphogenesis was markedly
perturbed by expression of FGF-10 in the fetal lung. In the postnatal
lung, induction of FGF-10 mRNA caused extensive multifocal, papillary,
and lepidic pulmonary adenomas lined by type II epithelial cells.
Tumors completely regressed in association with a rapid decrease in the
expression of type II cell markers after withdrawal of doxycycline. The
effects of FGF-10 were similar to, but distinct from, those of FGF-7 in
both fetal and postnatal lungs.
Effects of FGF-10 in lung morphogenesis.
In the present study, ectopic expression of FGF-10 in the respiratory
epithelium of fetal lung markedly perturbed lung morphogenesis and
caused dense, adenomatous malformations. The abnormalities were similar
to, but distinct from, those induced by FGF-7 (35, 40),
the latter causing generalized hyperplasia in the adult and marked
cystic changes in the fetal lung. Ultrastructural features of
respiratory epithelial cells in the tumors from FGF-10 transgenic mice
were characteristic of type II epithelial cells and stained intensely
for the differentiation markers TTF-1, proSP-C, and SP-B, consistent
with a role for FGF-10 in respiratory epithelial differentiation.
Targeted disruption of the mouse FGF-10 gene produced mice lacking both
limbs and lungs (25, 34). Taken together with the
observation that FGF-10 mRNA is produced by the lung mesenchyme at
restricted sites near branch sites of lung buds, FGF-10 signaling was
hypothesized to play a critical role in branching morphogenesis of the
lung. Considered in the light of failed lung formation in the FGF-10
gene-targeted mouse, the effects of ectopic expression of FGF-10
observed in the present study support the concept that precise
temporospatial control of FGF-10 expression is required for normal
branching morphogenesis.
Similar but distinct postnatal effects of FGF-10 and FGF-7.
Findings in the FGF-10 double-transgenic mice were distinct from those
in postnatal rodents treated with exogenous FGF-7 (41) or
in transgenic mice in which FGF-7 was expressed in the respiratory epithelium throughout lung development (35). In contrast
to the present findings in the FGF-10-expressing mice, organized tumor
formation was generally not seen in mice expressing FGF-7 in the
postnatal period (40). FGF-7 induced widespread
respiratory epithelial cell hyperplasia but did not cause
well-organized tumors. The finding that increased and ectopic
expression of FGF-10 mRNA in fetal lung produced dense, adenomatous
changes with mild cyst formation contrasted with the observations that
FGF-7 caused massive pulmonary cysts that were lethal to the fetus by
15-16 days of gestation (35, 44). These findings may
be related to differences in the extent and level of expression of the
FGFs or to distinct effects of the different polypeptides on pulmonary
cells; they may also indicate greater effects of FGF-7 on fluid and
electrolyte transport in the fetal lung. Increased staining for TTF-1
and surfactant proteins and infiltration by alveolar macrophages were observed in both FGF-7- and FGF-10-expressing mice. Whether the latter
finding represents a direct effect of the FGF family members on
macrophage migration and proliferation or represents a response to
increased cell turnover or tumor formation is unclear at present. The
observation that both FGF-7 and FGF-10 enhanced TTF-1 and surfactant
protein expression supports the concept that these polypeptides share
signaling pathways in respiratory epithelial cells.
The mechanisms underlying the observed differences in the effects of
FGF-7 and FGF-10 on pattern organization of lung tissues remain to be
clarified. Effects of FGF-10 on the fetal lung may be limited by as yet
unknown regulatory molecule(s) that limit synthesis, secretion, access,
or signaling by FGF-10 in a manner distinct from FGF-7. Because the
FGF-7 and FGF-10 transgenes are expressed in epithelial cells and not
in mesenchymal cells as in wild-type mice, bioavailability of the two
ligands or accessibility of the ligands to FGFR may be distinct in the
transgenic mice. Interaction of FGF-7 and FGF-10 with heparan sulfate
proteoglycans in the extracellular matrix or on the cell surface is
distinct, effects of FGF-10 being enhanced by heparin, whereas those of FGF-7 are inhibited (15), although the inhibitory effects
of heparin on the actions of FGF-7 are controversial (4).
Likewise, whereas FGF-7 and FGF-10 share binding to the FGFR2-IIIb
isoform, FGF-10 also binds to the FGFR1-IIIb isoform (21).
FGF-10 is sufficient to cause multifocal pulmonary tumors in the
postnatal lung.
Expression of FGF-10 caused multifocal, highly differentiated adenomas
in vivo, demonstrating that doxycycline-induced FGF-10 was sufficient
for production of pulmonary tumors. BrdU labeling was increased in
FGF-10-induced tumors, likely indicating its stimulatory effect on cell
proliferation. In the present study, FGF-10-induced tumors were
detected within 1-4 wk after treatment with doxycycline. In line
B, the tumors progressed rapidly, causing massive tumor
infiltration and respiratory distress. The extent of tumor formation
correlated in general with the sites and levels of transgene
expression. Despite multifocal tumors, much of the pulmonary parenchyma
remained unperturbed by expression of FGF-10 in line A mice.
This finding may be related to relatively more restricted sites of
expression of the transgene. Tumors from all FGF-10-expressing mice
consisted of highly differentiated pulmonary adenomas with
immunohistochemical and ultrastructural features of type II epithelial
cells. Staining for TTF-1, a homeodomain-containing transcription
factor critical to lung morphogenesis and gene expression (5,
16), was increased in the tumors, and withdrawal from doxycycline was associated with a rapid change in cell morphology and
immunostaining for TTF-1, proSP-C, and SP-B, all markers of type II
cell differentiation. Likewise, type II epithelial cell features were
noted in the hyperplastic epithelial cells in all of the tumors,
whether generated by the SP-C or CCSP promoters. The paucity of CCSP
staining in adult tumors suggests that FGF-10, even when expressed with
the CCSP promoter, may selectively influence respiratory epithelial
cell commitment or differentiation to type II epithelial cell subtypes.
The characteristics of immunostaining of surfactant proteins and TTF-1
observed in FGF-10-expressing mice were similar to those induced by
FGF-7 (35, 40).
Whether increased FGF signaling influences pulmonary
tumorigenesis to cause clinical disease is unclear. Altered FGFR and FGF production have been associated with oncogenesis in a variety of
organ systems. Non-small cell lung carcinomas, many with histological features similar to those presently observed in the FGF-10-expressing mice, represent an increasing subset of human lung cancers. Non-small cell tumors express FGFR, including FGFR1, FGFR2, FGFR3, and varying levels of FGF polypeptides, supporting their potential role in oncogenesis (3). In the present work, the tumors produced
in the FGF-10 double-transgenic mice regressed following withdrawal of
doxycycline; thus FGF-10 does not appear to be oncogenic during the
time period studied. Regression was associated with rapid loss of
cuboidal shape and markedly decreased expression of TTF-1, SP-B, and
proSP-C, consistent with a change in cell differentiation caused by
decreased FGF-10. TUNEL staining and electron microscopy did not
support changes consistent with apoptosis as a primary cause of
tumor regression after removal from doxycycline. Epithelial cells
detached from the basal lamina of the tumors after removal of
doxycycline, a process that may play a role in the resolution of the tumors.
Utility of the rtTA system for conditional expression in the lung
in vivo.
We recently generated permanent activator mouse lines that
express rtTA under control of the SP-C (expressed in distal bronchiolar and alveolar type II cells) and CCSP (expressed in nonciliated columnar
and alveolar respiratory epithelial cells) promoters (40).
In preliminary studies from this laboratory, induction of transgene
expression by doxycycline treatment of the dam was observed as early as
pc day 14 with the CCSP promoter and as early as pc
day 12.5 with the SP-C promoter (Perl, Tichelaar, and
Whitsett, unpublished observations). These observations are
consistent with previous studies demonstrating the expression of CCSP-
and SP-C-driven transgenes in the developing lung (10,
43). The finding that rtTA and FGF-10 transgenic mRNAs were
consistently detected in both conducting airways and alveolar type II
cells in CCSP-rtTA offspring was somewhat surprising and distinct from
the pattern of expression of other CCSP transgenes that generally
target only the conducting airways, an observation likely related to
positional effects modifying the sites of expression of CCSP-rtTA in
this mouse line. Differences in the levels and extent of transgene expression due to positional effects may also influence the phenotype in mouse lines generated for this study. The failure to detect FGF-10
mRNA in the absence of doxycycline in CCSP-rtTA × (teto)7CMV-FGF-10 activator mice demonstrates the
relatively tight regulation and cell specificity of this gene
expression system in vivo. Both SP-C-rtTA and CCSP-rtTA activator mice
induce target genes in a lung epithelial cell-restricted pattern under
control of doxycycline and should be useful for the study of pulmonary
development and function.
In conclusion, the present findings support the concept that
increased expression of a single gene, FGF-10, was sufficient to induce
organized tumors with type II epithelial cell characteristics in the
lung in vivo. The similar but also distinct effects of FGF-7 and FGF-10
on the prenatal and postnatal lung suggest that both shared a unique
pathway mediating FGF signaling by these two closely related ligands.
Present findings in the fetal and adult lung are consistent with the
previously proposed role for FGF-10 in directing respiratory cell
proliferation, pattern formation, and differentiation during branching
morphogenesis (2). FGF-10 enhanced expression of TTF-1 and
its regulatory targets, proSP-C and SP-B, in a reversible fashion,
supporting a critical role for FGF-10 in type II cell differentiation.
 |
ACKNOWLEDGEMENTS |
We acknowledge the excellent technical assistance of Paula Blair,
Lara Picard, Sherri Profitt, Jennifer Nation, and Ann Maher.
 |
FOOTNOTES |
The study was supported by the National Heart, Lung, and Blood
Institute Grants HL-56387 and HL-41496 and by the Cystic Fibrosis Research and Development Center from the Cystic Fibrosis Foundation.
Address for reprint requests and other correspondence: J. A. Whitsett, Div. of Neonatology and Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: jeff.whitsett{at}chmcc.org).
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.
Received 22 September 2000; accepted in final form 2 November 2000.
 |
REFERENCES |
1.
Arman, E,
Haffner-Krausz R,
Gorivodsky M,
and
Lonai P.
Fgfr2 is required for limb outgrowth and lung-branching morphogenesis.
Proc Natl Acad Sci USA
96:
11895-11899,
1999[Abstract/Free Full Text].
2.
Bellusci, S,
Grindley J,
Emoto H,
Itoh N,
and
Hogan BLM
Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung.
Development
124:
4867-4878,
1997[Abstract/Free Full Text].
3.
Berger, W,
Setinek U,
Mohr T,
Kindas-Mugge I,
Vetterlein M,
Dekan G,
Eckersberger F,
Caldas C,
and
Micksche M.
Evidence for a role of FGF-2 and FGF receptors in the proliferation of non-small cell lung cancer cells.
Int J Cancer
83:
415-423,
1999[ISI][Medline].
4.
Berman, B,
Ostrovsky O,
Shlissel M,
Lang T,
Regan D,
Vlodavsky I,
Ishai-Michaeli R,
and
Ron D.
Similarities and differences between the effects of heparin and glypican-1 on the bioactivity of acidic fibroblast growth factor and the keratinocyte growth factor.
J Biol Chem
274:
36132-36138,
1999[Abstract/Free Full Text].
5.
Bohinski, RJ,
DiLauro R,
and
Whitsett JA.
The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis.
Mol Cell Biol
14:
5671-5681,
1994[Abstract].
6.
Celli, G,
LaRochelle WJ,
Mackem S,
Sharp R,
and
Merlino G.
Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning.
EMBO J
17:
1642-1655,
1998[Abstract/Free Full Text].
7.
De Moerlooze, L,
Spencer-Dene B,
Revest J,
Hajhosseini M,
Rosewell I,
and
Dickson C.
An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signaling during mouse organogenesis.
Development
127:
483-492,
2000[Abstract/Free Full Text].
8.
Finch, PW,
Cunha GR,
Rubin JS,
Wong J,
and
Ron D.
Pattern of keratinocyte growth factor and keratinocyte growth factor receptors during mouse fetal development suggests a role in mediating morphogenic mesenchymal-epithelial interactions.
Dev Dyn
203:
223-240,
1995[ISI][Medline].
9.
Fu, Y-M,
Spirito P,
Yu Z-X,
Biro S,
Sasse J,
Lei J,
Ferrans VJ,
Epstein SE,
and
Casscells W.
Acidic fibroblast growth factor in the developing rat embryo.
J Cell Biol
114:
1261-1273,
1991[Abstract].
10.
Glasser, SW,
Korfhagen TR,
Wert SE,
Bruno MD,
McWilliams KM,
Vorbroker DK,
and
Whitsett JA.
Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice.
Am J Physiol Lung Cell Mol Physiol
261:
L349-L356,
1991[Abstract/Free Full Text].
11.
Gossen, M,
Freundlieb S,
Bender G,
Muller G,
Hillen W,
and
Bujard H.
Transcriptional activation by tetracyclines in mammalian cells.
Science
268:
1766-1769,
1995[ISI][Medline].
12.
Guo, L,
Degentein L,
and
Fuchs E.
Keratinocyte growth factor is required for hair development but not for wound healing.
Genes Dev
10:
165-175,
1996[Abstract].
13.
Horlick, RA,
Stack SL,
and
Cooke GM.
Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4.
Gene
120:
291-295,
1992[ISI][Medline].
14.
Hu, MC,
Qiu WR,
Wang Y,
Hill D,
Ring BD,
Scully S,
Bolon B,
DeRose M,
Luethy R,
Simonet WS,
Arakawa T,
and
Danilenko DM.
FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation.
Mol Cell Biol
18:
6063-6074,
1998[Abstract/Free Full Text].
15.
Igarashi, M,
Finch PW,
and
Aaronson SA.
Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7).
J Biol Chem
273:
13230-13235,
1998[Abstract/Free Full Text].
16.
Kimura, S,
Hara Y,
Pineau T,
Fernandez-Salguero P,
Fox CH,
Ward JM,
and
Gonzalez FJ.
The T/ebp null mouse: thyroid specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary.
Genes Dev
10:
60-69,
1996[Abstract].
17.
Klint, P,
and
Claesson-Welsh L.
Signal transduction by fibroblast growth factor receptors.
Front Biosci
4:
165-177,
1999.
18.
Korfhagen, TR,
Sheftelyevich V,
Burhans MS,
Bruno MD,
Ross GF,
Wert SE,
Stahlman MT,
Jobe AH,
Ikegami M,
Whitsett JA,
and
Fisher JH.
Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo.
J Biol Chem
273:
28438-28443,
1998[Abstract/Free Full Text].
19.
Korhonen, J,
Partanen J,
and
Alitalo K.
Expression of FGFR-4 mRNA in developing mouse tissues.
Int J Dev Biol
36:
323-329,
1992[ISI][Medline].
20.
Lin, S,
Na C,
Akinbi HT,
Apsley KS,
Whitsett JA,
and
Weaver TE.
Surfactant protein B (SP-B)-/- mice are rescued by restoration of SP-B expression in alveolar type II cells but not Clara cells.
J Biol Chem
274:
19168-19174,
1999[Abstract/Free Full Text].
21.
Lu, W,
Luo Y,
Kan M,
and
McKeehan WL.
Fibroblast growth factor-10. A second candidate stromal to epithelial cell andromedin in prostate.
J Biol Chem
274:
12827-12834,
1999[Abstract/Free Full Text].
22.
Luo, Y,
Lu W,
Mohamedali KA,
Jang JH,
Jones RB,
Gabriel JL,
Kan M,
and
McKeehan WL.
The glycine box: a determinant of specificity for fibroblast growth factor.
Biochemistry
37:
16506-16515,
1998[ISI][Medline].
23.
Mason, IJ,
Fuller-Pace F,
Smith R,
and
Dickson C.
FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial-mesenchymal interactions.
Mech Dev
45:
15-30,
1994[ISI][Medline].
24.
McKeehan, WL,
Wang F,
and
Kan M.
The heparan sulfate-fibroblast growth factor family: diversity of structure and function.
Prog Nucleic Acid Res Mol Biol
59:
135-176,
1998[ISI][Medline].
25.
Min, H,
Danilenko DM,
Scully SA,
Bolon B,
Ring BD,
Tarpley JE,
DeRose M,
and
Simonet WS.
FGF-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless.
Genes Dev
12:
3156-3161,
1998[Abstract/Free Full Text].
26.
Ohbayashi, N,
Hoshikawa M,
Kimura S,
Yamasaki M,
Fukui S,
and
Itoh N.
Structure and expression of the mRNA encoding a novel fibroblast growth factor, FGF-18.
J Biol Chem
273:
18161-18164,
1998[Abstract/Free Full Text].
27.
Orr-Urtreger, A,
Givol D,
Yayon A,
Yarden Y,
and
Lonai P.
Developmental expression of two murine fibroblast growth factor receptors, flg and bek.
Development
113:
1419-1434,
1991[Abstract].
28.
Park, WY,
Miranda B,
Lebeche D,
Hashimoto G,
and
Cardoso WV.
FGF-10 is a chemotactic factor for distal epithelial buds during lung development.
Dev Biol
201:
125-134,
1998[ISI][Medline].
29.
Peters, K,
Werner S,
Liao X,
Wert S,
Whitsett J,
and
Williams L.
Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung.
EMBO J
13:
3296-3301,
1994[Abstract].
30.
Peters, KG,
Werner S,
Chen G,
and
Williams LT.
Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse.
Development
114:
233-243,
1992[Abstract].
31.
Powell, PP,
Wang CC,
Horinouchi H,
Shepherd K,
Jacobson M,
Lipson M,
and
Jones R.
Differential expression of fibroblast growth factor receptors 1 to 4 and ligand genes in late fetal and early postnatal rat lung.
Am J Respir Cell Mol Biol
19:
563-572,
1998[Abstract/Free Full Text].
32.
Reichman-Fried, M,
Dickson B,
Hafen E,
and
Shilo BZ.
Elucidation of the role of breathless, a Drosophila FGF receptor homolog, in tracheal cell migration.
Genes Dev
8:
428-439,
1994[Abstract].
33.
Sannes, PL,
Burch KK,
and
Khosla J.
Immunohistochemical localization of epidermal growth factor and acidic and basic fibroblast growth factors in postnatal developing and adult rat lungs.
Am J Respir Cell Mol Biol
7:
230-237,
1992[ISI][Medline].
34.
Sekine, K,
Ohuchi H,
Fujiwara M,
Yamasaki M,
Yoshizawa T,
Yagishita N,
Matsui D,
Koga Y,
Itoh N,
and
Kato S.
FGF10 is essential for limb and lung formation.
Nat Genet
21:
138-141,
1999[ISI][Medline].
35.
Simonet, WS,
DeRose ML,
Bucay N,
Nguyen HQ,
Wert SE,
Zhou L,
Ulich TR,
Thomason A,
Danilenko DM,
and
Whitsett JA.
Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.
Proc Natl Acad Sci USA
92:
12461-12465,
1995[Abstract].
36.
Stripp, BR,
Sawaya PL,
Luse DS,
Wikenheiser K,
Wert S,
Huffman JA,
Lattier DL,
Singh G,
Katyal SL,
and
Whitsett JA.
Cis-acting elements that confer lung epithelial cell expression of the CC10 gene.
J Biol Chem
267:
14703-14712,
1992[Abstract/Free Full Text].
37.
Sutherland, D,
Samakoulis C,
and
Krasnow MA.
Branchless encodes a Drosophila Fgf homolog that controls tracheal migration and the pattern of branching.
Cell
87:
1091-1101,
1996[ISI][Medline].
38.
Szebenyi, G,
and
Fallon JF.
Fibroblast growth factors as multifunctional signaling factors.
Int Rev Cytol
185:
45-106,
1999[ISI][Medline].
39.
Tagashira, S,
Harada H,
Katsumata T,
Itoh N,
and
Nakatsuka M.
Cloning of mouse FGF-10 and up-regulation of its gene expression during wound healing.
Gene
197:
399-404,
1997[ISI][Medline].
40.
Tichelaar, JW,
Lu W,
and
Whitsett JA.
Conditional expression of fibroblast growth factor-7 in the developing and mature lung.
J Biol Chem
275:
11858-11864,
2000[Abstract/Free Full Text].
41.
Ulich, TR,
Watson LR,
Yin S,
Guo K,
Wang P,
Thang H,
and
del Castillo J.
The intratracheal administration of endotoxin and cytokines.
Am J Pathol
138:
1485-1496,
1991[Abstract].
42.
Weinstein, M,
Xu X,
Ohyama K,
and
Deng CX.
FGF-R-3 and FGF-R-4 function cooperatively to direct alveogenesis in murine lung.
Development
125:
3615-3623,
1998[Abstract/Free Full Text].
43.
Wert, SE,
Glasser SW,
Korfhagen TR,
and
Whitsett JA.
Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.
Dev Biol
156:
426-443,
1993[ISI][Medline].
44.
Zhou, L,
Graef RW,
McCray PB,
Simonet WS,
and
Whitsett JA.
Keratinocyte growth factor stimulates CFTR-independent fluid secretion in the fetal lung in vitro.
Am J Physiol Lung Cell Mol Physiol
271:
L987-L994,
1996[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 280(4):L705-L715
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