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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 |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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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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 |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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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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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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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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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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 |
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We acknowledge the excellent technical assistance of Paula Blair, Lara Picard, Sherri Profitt, Jennifer Nation, and Ann Maher.
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FOOTNOTES |
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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.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Dox). Line B mice were treated for 12 days (+Dox) or
for 12 days followed by 6 days without doxycycline (+Dox/
