From the Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
Received for publication, August 14, 2002, and in revised form, September 30, 2002
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ABSTRACT |
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Although fibroblast growth factor (FGF)
signaling is required for the formation of the lung in the embryonic
period, it is unclear whether FGF receptor activity influences lung
morphogenesis later in development. We generated transgenic mice
expressing a soluble FGF receptor (FGFR-HFc) under conditional control
of the lung-specific surfactant protein C promoter (SP-C-rtTA), to inhibit FGF activity at various times in late gestation and
postnatally. Although expression of FGFR-HFc early in development
caused severe fetal lung hypoplasia, activation of the transgene in the
postnatal period did not alter alveolarization, lung size, or
histology. In contrast, expression of the transgene at post-conception
day E14.5 decreased lung tubule formation before birth and caused severe emphysema at maturity. FGFR-HFc caused mild focal emphysema when
expressed from E16.5 but did not alter alveolarization when expressed
after birth. Although FGF signaling was required for branching
morphogenesis early in lung development, postnatal alveolarization was
not influenced by FGFR-HFc.
The lung buds evaginate from the foregut endoderm and undergo
stereotypic branching to form conducting and peripheral airspaces critical for gas exchange after birth. In humans and mice, the alveoli
are formed primarily in the postnatal period, during which the numbers
of alveoli increase and the alveolar capillary gas exchange area
expands. Lung morphogenesis is dependent upon autocrine-paracrine signaling between the splanchnic mesenchyme and the endodermally derived lung buds that form the developing respiratory epithelium. A
number of growth factors and transcription factors, including fibroblast growth factors
(FGFs),1 plays important
roles during lung morphogenesis, regulating cell migration,
proliferation, and differentiation (Refs. 1-3 for review).
Fibroblast growth factors (FGFs) comprise a family of low molecular
weight polypeptides that are involved in the morphogenesis of many
organs, including the lung (4). At least seven FGFs are expressed in
the developing lung, including aFGF, bFGF, FGF-7, FGF-9, FGF-10, and
FGF-18 (5-12). Likewise, all of the known FGF receptors
(FGFR1-5) are expressed in the lung (13-18). The extracellular domains of the FGFR consist of three immunoglobulin-like domains (D1, D2, and D3). Ligand binding is
mediated by the C-terminal portion of the D3 domain.
Classification of the FGFRs is dependent upon differences in the
D3 domain. Three discrete subgroups of D3
domains have been identified, each representing variably spliced isoforms (4). Although all FGFRs are expressed in fetal and postnatal
lung, the temporal-spatial distribution of each receptor and ligands
varies. FGFR2 is restricted to the epithelial cells of the developing
lung buds at the onset of lung organogenesis at E9.5 (19). The FGFR2
becomes increasing restricted to the peripheral lung buds during
branching morphogenesis. FGFR2IIIb is expressed in respiratory
epithelial cells, whereas FGFR2IIIc is distributed in mesenchymal cells
of the lung (16, 19, 20).
The important role of FGF signaling during formation of the lung was
revealed by both in vitro and in vivo
experiments. FGF-7 caused cystic dilation of the fetal lung and cell
proliferation in the postnatal lung (21, 22). A number of in
vitro and in vivo experiments support the concept that
FGF-10 production by lung mesenchyme is critical for early lung
morphogenesis. FGF-10 gene-targeted mice failed to develop limbs or
lungs (23, 24). Likewise, FGF-10 enhanced cell proliferation and
chemotaxis of respiratory epithelial cells of the fetal lung in
vitro (7, 25). The finding that FGFR2b gene-targeted mice had a
phenotype similar to that seen in the FGF-10 ( Because deletion of either FGF-10 or inhibition of FGFR2 activity
blocked lung formation, FGF signaling plays a critical role in early
lung morphogenesis. However, because lung formation fails to occur in
these gene-targeted mice, it is unclear if FGF receptor signaling is
required only for the initiation of lung morphogenesis or functions
later in development. Because most of lung growth and alveolarization
occurs postnatally, it is not known whether FGF signaling plays a role
after the embryonic period and whether the postnatal emphysema seen in
the FGFR3/4 ( Transgene Construct--
FGFR-HFc consists of the extracellular
domain of mouse FGFR2b and the heavy chain hinge and Fc domain of mouse
immunoglobulin (28). FGFR-HFc cDNA was inserted between the
(tetO)7-CMV minimal promoter and the 3'-untranslated region
of the bovine growth hormone gene as previously described (22, 30) (see
Fig. 1).
Transgenic Mouse Lines--
Transgenic mouse lines bearing the
(tetO)7-CMV-FGFR-HFc were established in the FVB/N strain
after oocyte microinjection of the (tetO)7-CMV-FGFR-HFc
construct. Heterozygous (tetO)7-CMV-FGFR-HFc mice were
viable and had no malformations. Five independent mouse lines bearing
the FGFR-HFc were generated. Mice transmitting the (tetO)7-CMV-FGFR-HFc were mated with SP-C-rtTA mice that
express the rtTA activator under control of the human 3.7-kb SP-C
promoter, selectively directing expression of transgenes in respiratory epithelial cells of the lung (22, 30). Transgenic mice were identified
by using PCR primers specific for each transgenes. Severe lung
hypoplasia was observed in double transgenic offspring from five
independent founder lines when the dam was maintained on doxycycline
throughout pregnancy. Transgenic line 5.9 was then chosen for further analysis.
Animal Use and Doxycycline Administration--
Transgenic mice
were kept in the pathogen-free vivarium according to
institutional guidelines. Gestation was estimated by the detection of
vaginal plug and correlated with average fetal weight at the time of
sacrifice. Doxycycline was administered in the drinking water at a
final concentration of 1 mg/ml or in the food pellets at concentration
of 25 mg/g (Harlan Teklar, Madison, WI) for described time
periods. Drinking water that contained doxycycline was changed three
times per week because of light sensitivity. Activity of the
doxycycline in the food pellets was stable for up to 6 months.
Tissue Preparation, Histology, and
Immunohistochemistry--
Mice were killed by lethal injection of
anesthetic reagent (ketamine, xylasine, and acepromasine). Lungs were
inflation-fixed with 4% paraformaldehyde in PBS at 25-cm water
pressure and immersed in same fixative. Tissue was fixed overnight,
washed with PBS, dehydrated through a series of ethanol, and embedded
in paraffin. Tissue sections were stained with hematoxylin-eosin,
orcein, or trichrome for histology. Immunohistochemistry for thyroid
transcription factor-1 (TTF-1) (Dako Corp., Carpenteria, CA), mature
surfactant protein-B (SP-B) (AB3426, Chemicon, Inc., Temecula, CA), and
platelet endothelial cell adhesion molecule-1 (PECAM-1) were performed as previously described (31).
Quantitation of Pulmonary Vessels--
Blood was flushed from
the lungs by infusion of heparinized saline through a catheter in the
main pulmonary artery. A barium gelatin heated at 70 °C was injected
at 70 mmHg pressure until the vessels were filled uniformly filled with
barium. Lungs were then inflation fixed with 4% paraformaldehyde in
PBS at 25-cm water pressure. Tissues were fixed overnight and washed
with PBS and dehydrated through a series of ethanol. The left lobe was excised and embedded in paraffin. Sections were stained with
hematoxylin-eosin and barium-filled pulmonary arteries counted. At
least 12 sections were counted per animal. Lung fields containing large
airways or major vessels were excluded.
Proliferation Index--
Animals were injected intraperitoneally
with bromodeoxyuridine (BrdUrd, 1 mg/g weight) and killed 2 h thereafter. BrdUrd-labeled cells were detected by immunostaining
using mouse monoclonal antibody in accordance with the manufacturer's
instruction (Zymed Laboratories, Inc., San Francisco, CA). Cells were
counted in randomly selected fields, n = 4 animals per group.
RT-PCR--
Lung tissues were homogenized in TRIzol reagent
(Invitrogen, Gaithersburg, MD), and RNA were extracted according to the
manufacturer's instructions. RNA was treated with DNase prior to
cDNA synthesis. RNA (5 µg) was reverse-transcribed and analyzed
by PCR for FGFR-HFc mRNA. Primers for the FGFR-HFc were
designed as follows: The forward primer was located in the coding
region of extracellular domain of FGFR2 (5'-CAG GCC AAC CAG TCT GCC TGG
C-3'), and the reverse primer was located in the coding region of the
Fc domain of immunoglobulin (3'-CCT CCA CGT GTG TCG AGT CTG C-5').
Surfactant proteins A, B, C, and D cDNA were quantitated by
real-time PCR of whole lung cDNA after optimization of primers and
conditions using the Smart Cycler®. The mRNA levels were
normalized to Lung Explant Cultures--
Lung buds were isolated from mouse
embryos at E11-13.5 obtained from the breeding of SP-C-rtTA and
(tetO)7-CMV-FGFR-HFc mice. Lung buds were cultured with or
without doxycycline (1 µg/ml) on 13-mm diameter, 8.0-µm pore size
Track-Etch membranes (Whatman 110414). Each well contained 1 ml of
Dulbecco's modified Eagle medium. Human recombinant FGF-7 (Pepro Tech
Inc., Rocky Hill, NJ) was diluted in PBS and added to each well at
final concentrations of 0.625-20 ng/ml. Lung buds were grown at
37 °C in a humidified 5% CO2-95% air incubator.
Photographs were taken with a stereoscopic microscope.
Conditional Expression of FGFR-HFc--
Double and single
transgenic mice of all genotypes survived normally and no abnormalities
in lung histology were observed in the absence of doxycycline. To
determine the effects of doxycycline on the expression of FGFR-HFc,
adult SP-C-rtTA, (tetO)7-CMV-FGFR-HFc double transgenic and
single transgenic mice were treated with doxycycline for 3 days.
FGFR-HFc RNA was detected only in double transgenic mice treated with
doxycycline (Fig. 1B). When
the dams were maintained on doxycycline from conception, severe lung
hypoplasia was observed in all double transgenic pups at E18,
demonstrating that the expression of the FGFR-HFc markedly abrogated
lung morphogenesis during the embryonic period (Fig.
2).
Prenatal Expression of FGFR-HFc Causes Emphysema--
To determine
the temporal requirements for FGF signaling during lung morphogenesis,
the dams or pups were treated with doxycycline at various developmental
time periods (Fig. 3A). When
pups were treated from PN0 to PN25, no abnormalities in lung growth or
histology were observed (Fig. 3, A and E). Double
transgenic mice treated with doxycycline from E14.5 developed severe,
diffuse emphysema when assessed at PN25 or at 6 weeks of age (Fig. 3,
B and F) and data not shown. In contrast, less
extensive, focal emphysema was observed when the mice were exposed to
doxycycline from E16.5 to PN25 (Fig. 3, C and G).
The severity of emphysema was similar in double transgenic mice treated
from E14.5 to PN0 (removed from doxycycline thereafter) (Fig. 3,
D and H) and those treated with doxycycline from
E14.5 until killing at PN25 (Fig. 3, B and F). After treatment from E14.5, percent airspace, determined by lung morphometry, was significantly increased (78.2 ± 1.9, mean ± S.E., n = 5, p < 0.006) compared
with controls (69.9 ± 0.69, n = 4) and was less
affected when the mice were treated from E16.5 (74.5 ± 1.7, p = 0.08, n = 5) by ANOVA when assessed
at PN25. Lung pathology consisted of airspace enlargement without
evidence of inflammation, cellular infiltrates, or fibrosis (as
assessed by trichrome and orcein staining, data not shown), suggesting
that the lesions represent abnormalities in morphogenesis rather than
inflammatory remodeling. At E18.5, the lung weight to body weight ratio
was decreased when dams were treated from E14.5-18.5 but was not
altered when assessed at PN25 (Table
I).
Maintenance of Cell Differentiation--
To assess whether
FGFR-HFc altered epithelial or vascular differentiation, lung sections
from double transgenic mice that were treated from E14.5 to PN25 were
stained with antibody against mature SP-B, TTF-1, and/or PECAM,
respiratory epithelial and pulmonary vascular cell markers,
respectively (Figs. 4 and
5). Although the numbers of alveoli and
peripheral vessels were decreased, there were no differences in the
intensity of staining for PECAM (Fig. 4, C and
F). The proportion of SP-B-positive cells in the peripheral
lung were significantly decreased in double transgenic mice (Figs. 4
and 5). Morphometric analysis and pulmonary arteriograms demonstrated
decreased pulmonary vessel density, consistent with emphysema. A
significant reduction in pulmonary vessels was observed in adult mice
that had been treated with doxycycline from E14.5 to 6 weeks of age
(Fig. 6, A and B).
Despite the loss of pulmonary vascularity, combined and right
ventricular weights were not different in these mice at 8 months of
age. TTF-1 was detected in the nuclei of respiratory epithelial cells
in both transgenic mice and controls (Fig. 4, B and
E).
Inhibition of Lung Morphogenesis by FGFR-HFc before Birth--
To
determine the timing of events involved in the perturbation of lung
morphogenesis following exposure to doxycycline, dams were maintained
on doxycycline from E14.5 and the lung tissue evaluated at E16.5 (Fig.
7) and E18.5 (Fig.
8). On embryonic day 16.5, the numbers of
respiratory tubules were reduced in lungs of the double transgenic
fetuses (Fig. 7). No differences in the intensity of TTF-1
immunostaining or BrdUrd labeling (Figs. 7 and 8) were observed. At
E18.5, numbers of respiratory saccules were significantly decreased in
the double transgenic mice (Fig. 8). Airspaces were larger than those
seen in control fetuses (Fig. 8, A and D). The
proliferation index, as assessed by BrdUrd labeling, was reduced at
E18.5 (p < 0.01) (Fig.
9). However, no statistical differences were observed in the
proliferation index on E16.5 despite the arrest of sacculation observed
histologically (Figs. 7 and 9). There was no evidence of cell necrosis,
nuclear fragmentation, or condensation, suggesting that widespread cell
injury or apoptosis did not account for the observed emphysema or
lung hypoplasia.
FGFR-HFc Did Not Alter Surfactant Protein and TTF-1
mRNAs--
Surfactant protein mRNAs were quantitated by S1
nuclease assay on E16.5 and E18.5 after treatment with doxycycline from
E14.5. At E16.5, SP-B mRNA was decreased in double transgenic mice.
No significant differences in SP-B mRNA were detected on E18.5.
Quantification of TTF-1 mRNA in PN25-old lungs after treatment with
doxycycline also revealed no differences (data not shown).
FGFR-HFc Inhibits Branching Morphogenesis and Responses to FGF-7 in
Vitro--
Lung explants from E11.5 embryos were cultured in the
presence and absence of doxycycline (Fig.
10). Doxycycline did not alter lung
growth or branching in controls. In contrast, lung branching was not
altered in tissue from double transgenic mice in the absence of
doxycycline (data not shown). Branching of lung tubules from double
transgenic mice was inhibited by doxycycline (1 µg/ml) (Fig. 10). To
assess whether FGFR-HFc inhibited responses to ectopic FGF-7 in
vitro, lung explants from double transgenic mice (E13.5) were
treated with FGF-7 in the presence of doxycycline (Fig.
11). FGF-7 caused cystic dilation of
the lung buds in control tissues, as previously described (21). In
contrast, doxycycline inhibited cyst formation in the lungs from the
double transgenic pups in vitro, demonstrating the ability
of the FGFR-HFc to block FGF-7-induced swelling (Fig. 11).
Conditional expression of FGFR-HFc in respiratory epithelial cells
of the developing fetal lung caused emphysema in the postnatal period.
The effects of FGFR-HFc were time-dependent. Severe,
permanent emphysema was observed in mature mice when the transgene was
activated from E14.5 to birth. Milder lung abnormalities were noted
when expressed from E16.5 to birth and thereafter. The effects of
FGFR-HFc during late fetal lung development contrasted with the lack of effects of the transgene when expressed only in the postnatal period.
Because most of the lung growth and alveolarization occur postnatally,
most pulmonary morphogenesis occurs by pathways that are not influenced
by the FGFR-HFc transgene.
Once established, emphysema persisted despite the removal of the mice
from doxycycline, demonstrating the irreversibility of the
architectural abnormalities. Emphysema in the FGFR-HFc transgenic mice
was not associated with inflammation, fibrosis, or cellular
infiltrates, consistent with defects in lung morphogenesis rather than
inflammation. Likewise, expression of the FGFR-HFc did not cause
necrosis or cell injury, and there was no evidence of nuclear
fragmentation. Taken together, these findings support the concept that
FGFR-HFc inhibited expansion of precursor cells that require FGF
signaling in the pseudoglandular-canalicular period of development
(E12.5-E16.5). Thereafter, lung morphogenesis proceeded relatively
normally despite the expression of the FGF receptor inhibitor.
The FGFR-HFc is a known inhibitor of FGF signaling, binding various FGF
ligands, including FGF-10, FGF-1, and FGF-7. Widespread expression of
this chimeric gene under control of the murine mammary tumor virus
promoter caused abnormalities in the skeleton, limbs, skin, kidney,
endocrine organs, and lung (28). Because FGFR2IIIb and FGF-10 are both
required for early lung morphogenesis, the presently
observed effects of the FGFR-HFc are consistent with inhibition of FGF
signaling in epithelial cells of the peripheral lung tubules during
their formation. The SP-C promoter is highly active in the fetal and
postnatal respiratory epithelium, directing expression of transgenes in
a temporal-spatial pattern similar to that of the FGFR2 (32). Because
inhibition of lung morphogenesis was observed when the SP-C promoter
was used to express a membrane-associated FGFR2 mutant, the present
findings are consistent with the requirement of FGF signaling in a
subset of respiratory epithelial cells that are critical for
morphogenesis of the peripheral lung. Interestingly, in FGFR-HFc, FGFR2
mutant-expressing and FGF-10 ( In contrast to the severe effects of FGFR-HFc on fetal lung
formation, postnatal alveolarization proceeded independently of the
expression of the transgene. This result is not likely related to
developmental changes in the expression of the
SP-C-dependent transgene, which is known to be expressed at
high levels in the postnatal period (32, 35). The SP-C promoter is
increasingly active in late gestation and in the postnatal period. SP-C
promoter-driven rtTA was expressed throughout the peripheral lung
prenatally and perinatally in the SP-C-rtTA line used in the present
study (32, 35). Previous in situ hybridization studies
utilizing this same SP-C-rtTA transgenic line with several
(tetO)7 target constructs demonstrated that target gene
expression was readily detected and is highly inducible, the latter
being expressed in subsets of acinar and alveolar regions of the
postnatal lung (22, 30, 35). In the present study, FGFR-HFc RNA was
readily detected in the adult lung after exposure to doxycycline. Thus
the lack of effect of FGFR-HFc on alveolarization in the postnatal
period is not likely related to changes in expression of the transgene but to the lack of a requirement for FGF signaling after birth.
Despite the emphysema, proximal-distal patterning of respiratory
epithelial cell morphology and mRNAs were maintained in the FGFR-HFc-expressing mice. Both TTF-1 and SP-B mRNAs were expressed at sites similar to these observed in control mice. The emphysematous lungs of the affected mice contained fewer type II epithelial cells and
pulmonary vessels, findings likely related to airspace enlargement.
Although the lung-body weight ratio was reduced at E18.5 in
FGFR-HFc-expressing mice, the ratio was not different at PN25, despite
the presence of severe emphysema. This observation may indicate that
compensatory growth has occurred during the postnatal period. The
relative content of SP-B and TTF-1 mRNAs was similar at E18.5 and
thereafter. Maintenance of SP-B expression is consistent with postnatal
survival of the FGFR-HFc-expressing mice, because severe deficiency of
SP-B causes lethal respiratory distress at birth (26).
Not surprisingly, abnormalities of alveolar structures were
associated with decreased vascularity. Morphometric analysis
demonstrated decreased numbers of peripheral blood vessels seen after
barium labeling of whole mount preparations. Thus, the paucity of
endodermally derived respiratory epithelial cells was correlated with a
failure to form vascular tissues, perhaps mediated by decreased
production of angiogenic and vasculogenic factors. It is also possible
that the FGFR-HFc directly inhibited FGF signaling to alter vascular development.
In summary, these studies demonstrate a precise temporal requirement
for FGF signaling for formation of the lung during the pseudoglandular-canalicular period of development. Although inhibition of FGF signaling during this period caused irreversible emphysema, postnatal alveologenesis proceeded normally despite expression of the
FGFR inhibitor. In human infants, lung hypoplasia is associated with a
number of clinical syndromes, including oligohydramnios, renal
agenesis, and diaphragmatic hernia. The severity of lung hypoplasia and
respiratory dysfunction in these syndromes is strongly influenced by
the timing of the initiating events, the human lung being most
vulnerable during the canalicular period of development. These clinical
observations and the present transgenic model support the concept that
perturbation of progenitor cells at critical times during lung
morphogenesis can cause irreversible emphysema in the postnatal period.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES
/
) mice suggests that
FGF-10 binds and activates the FGFR2 receptor (20). Expression of
either Sprouty-2 or a fusion protein consisting of the
FGFR-D2 and D3 domains and the mouse
immunoglobulin Fc fragment blocked lung morphogenesis by inhibiting FGF
signaling in vivo (27, 28). Taken together, FGF-10 and
FGFR2IIIb are required for early lung-branching morphogenesis. In
contrast, double gene targeting of FGFR3 and FGFR4 did not alter
prenatal lung development but caused defects in alveolarization in the
postnatal lung (29).
/
) mice is mediated by events occurring prenatally. To
assess the role and timing of FGF signaling in perinatal and postnatal
lung morphogenesis, we generated transgenic mice that express a soluble
dominant-negative FGFR2 (FGFR-HFc) under conditional control of
doxycycline using the reverse tetracycline transactivator system.
Inhibition of FGF signaling in the late gestation, but not postnatally,
inhibited cell proliferation and caused emphysema that was evident at maturation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-actin mRNA.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Transgenic constructs. A, the
structures of the mouse FGFR2b and FGFR-HFc are represented
(a and b). The D2 and D3
Ig-like domains of the FGFR2b are fused with the mouse Ig heavy chain
hinge and Fc domains. FGFR-HFc cDNA was cloned between the CMV
minimal promoter and bovine growth hormone polyadenylation signal
(bGH pA) (c). In the activator mice, the reverse
tetracycline transactivator (rtTA) was cloned 3' to the
human 3.7-kb SP-C (hSP-C) promoter (d).
B, RNA was extracted from the lungs of adult mice treated
with doxycycline for 3 days. -Actin and FGFR-HFc mRNAs were
identified by RT-PCR after gel electrophoresis. cDNA synthesis was
performed in the presence or absence of reverse transcriptase, and no
bands were seen in the absence of RT (data not shown).
(tetO)7-FGFR-HFc single transgenic mouse (lane
1), SP-C-rtTA plus (tetO)7-FGFR-HFc double transgenic
mice (lanes 2 and 3). Mice were treated with
doxycycline (lanes 1 and 2) or untreated
(lane 3).
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Fig. 2.
Effect of FGFR-HFc on lung
morphogenesis. Dams were treated with doxycycline from E6.5
to E18.5 and killed at E18.5. Lungs from FGFR-HFc-expressing mice were
severely hypoplastic, lacking most of the lung periphery
(B). The figure is representative of at least 10 affected
pups. Bars represent 2 mm.
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Fig. 3.
Time-dependent effects of the
FGFR-HFc. Periods of treatment with doxycycline are shown
(A). Lung sections were stained with hematoxylin-eosin
(B). Single transgenic or wild type littermates were treated
as described in A and analyzed at PN25. Roman
numerals refer to the period of doxycycline treatment
(A). Double transgenic mice expressing FGFR-HFc developed
emphysema when exposed to doxycycline in the prenatal but not postnatal
period. Bar = 100 µm. The figures are representative
of at least n = 4 mice per group.
Effect of FGFR-HFc on lung body weight ratio at E18.5
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Fig. 4.
Immunohistochemistry for SP-B, TTF-1, and
PECAM-1 on PN25 lungs. Lung sections were prepared on PN25.
FGFR-HFc-expressing (D-F) and controls (A-C)
mice were maintained on doxycycline from E14.5 to PN25. Lung sections
were stained with an antibody against mature SP-B (A and
D), TTF-1 (B and E), or PECAM-1
(C and F). Bar = 100 µm. The
figures are representative of at least n = 4 per
group.
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Fig. 5.
Emphysema and decreased numbers of type II
epithelial cells in FGFR-HFc-expressing mice. Mice were treated
with doxycycline from E14.5 to PN25. Lungs from control (A)
or FGFR-HFc-expressing (B) mice were stained for SP-B.
Bars = 100 µm. Numbers of SP-B staining and total
cells were counted (C). Five different areas were randomly
selected, comparing n = 5 animals in each group.
Differences (mean ± S.E.) were assessed by Student's
t test. The proportion of SP-B-staining cells was
significantly decreased.
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Fig. 6.
Effect of FGFR-HFc on blood vessel
density. Lung sections were prepared from PN25 control or
FGFR-HFc-expressing mice, treated with doxycycline from E14.5 to PN25.
After barium injection, lungs were stained and imaged (A and
B). Bar = 100 µm. Numbers of vessels
filled with barium were compared. Five different areas were randomly
selected from three mice from each group (B). Differences
(mean ± S.E.) were assessed by Student's t
test.
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Fig. 7.
Effect of FGFR-HFc on lung histology at
E16.5. Dams were treated with doxycycline from E14.5, and sections
were prepared at E16.5. Tissues were stained with H&E (A and
D), immunostained for TTF-1 (B and E),
or labeled with BrdUrd and stained to assess cellular proliferation
(C and F). The numbers of small tubules were
diminished in FGFR-HFc-expressing mice compared with controls.
Intensity and distribution of TTF-1 staining were not changed. No
differences in the numbers of BrdUrd-positive cells were observed
(C and F). Bar = 100 µm.
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Fig. 8.
Effect of FGFR-HFc on lung histology at
E18.5. Dams were treated with doxycycline from E14.5, and lung
sections were prepared at E18.5. Tissues were stained by H&E
(A and D), immunostained for TTF-1 (B
and E), or labeled with BrdUrd (C and
F) and stained to assess cellular proliferation. Air spaces
of FGFR-HFc-expressing mice were larger than controls. Numbers of lung
tubules were decreased in FGFR-HFc mice. No differences in the
distribution of TTF-1 immunostaining were observed. The numbers of
BrdUrd-stained cells were reduced in FGFR-HFc mice. Bar = 100 µm.
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Fig. 9.
Effect of FGFR-HFc on BrdUrd labeling.
Control or FGFR-HFc-expressing mice were maintained on doxycycline from
E14.5 and analyzed at E16.5 and E18.5. The dams were injected with
BrdUrd, and the pups were killed 2 h later. Tissues were
immunostained for BrdUrd. Data from five different sections were
counted from n = 4 mice per group. Differences
(mean ± S.E.) were assessed by Student's t
test.
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Fig. 10.
FGFR-HFc inhibited lung branching in
vitro. Fetal lung explants were placed in tissue culture at E11.5.
Lungs were cultured in Dulbecco's modified Eagle medium containing
doxycycline (1 µg/ml) and imaged at 0-50 h. Extensive branching was
noted in controls (A, C, E, and
G). Branching was inhibited in lungs from double transgenic
mice (B, D, F, and H).
Bar = 1 mm.
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Fig. 11.
FGFR-HFc-inhibited effects of FGF-7 in
vitro. Fetal lung explants were placed in tissue
culture at E13.5. Lungs were cultured in media containing doxycycline
(1 µg/ml). FGF-7 was added (15 ng/ml) after 12 h of culture.
Tissue was imaged from 0 to 60 h. Controls were maintained without
(A, D, G, and J) or with
(B, E, H, and K) FGF-7.
Double transgenic mice were treated with FGF-7 (C,
F, I, and L). Bar = 1 mm.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
/
) gene-targeted mice, some elements
of the proximal trachea were formed (23, 28, 33). In contrast, deletion
of FGFR2IIIb resulted in failure of formation of trachea and main stem
bronchi (20). In the present study, maintenance of trachea and main
bronchi may indicate that their development is independent of the
FGFR-HFc or that the SP-C promoter was not active in the proximal
regions of the forming lung buds. Thus, some aspects of proximal lung
tubule formation may be independent of FGF-10 but dependent on other
FGF signaling, perhaps mediated by other FGF ligands. Recent cell
lineage studies demonstrated that proximal and peripheral airways are
distinguished early in embryogenesis and that the members of progenitor
cells forming the lung periphery were highly restricted at the tips of
main bronchi at E12.5-13.5 (34). The present findings support the
concept that FGF signaling to this subset of precursors is required
between E12.5 and E14.5 for their survival and expansion during
formation of the lung periphery, with their loss causing severe lung
hypoplasia. We hypothesize that partial loss of these progenitors later
(E14.5-E16.5) results in a partial loss of progenitor cells and
resultant emphysema. Thus, FGF activity may be required at precise
times for survival and/or expansion of only subsets of progenitor cells
that are critical for the formation of the peripheral lung.
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ACKNOWLEDGEMENTS |
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The FGFR-HFc cDNA was kindly provided by William LaRochelle. We thank Tim Le Cras for technical assistance with barium infusion of the pulmonary arteries.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL56387 and Fonds zur Förderung der Wissenschaftlichen Forschung Schrödinger Grant J1959-GEN.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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Cincinnati Children's Hospital Medical Center, Divisions of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-636-4830; Fax: 513-636-7868; E-mail: jeff.whitsett@chmcc.org.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M208328200
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; FGFR-HFc, transgenic mice expressing a soluble FGF receptor; CMV, cytomegalovirus; PBS, phosphate-buffered saline; TTF-1, thyroid transcription factor-1; SP-B and -C, surfactant proteins B and C; PECAM-1, platelet endothelial cell adhesion molecule-1; BrdUrd, bromodeoxyuridine; RT, reverse transcriptase; E, embryonic day; PN, postnatal day; rtTA, reverse tetracycline transactivator.
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