1 Department of Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60607-7170; and 2 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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Previously, we showed
that newborn forkhead box (Fox)f1(+/) mice with diminished
pulmonary FoxF1 levels died of severe lung hemorrhage and exhibited
abnormal formation of alveolar sacs and capillaries. Another group
recently reported that Foxf1(+/
) mouse embryos displayed a
number of organ and skeletal defects including fusion of lung lobes.
However, identification of pulmonary genes whose altered expression was
associated with the lobular fusion defect in Foxf1(+/
)
lungs remains uncharacterized. The present study was conducted to
determine the nature of the malformations leading to lung fusions in
the FoxF1 embryos and to identify potential signaling pathways
influenced by FoxF1 haploinsufficiency. We show that
Foxf1(+/
) embryos exhibit defects in formation and branching of primary lung buds, which causes fusion of the right lung
lobes and vessels. The severity of the Foxf1(+/
) lung
fusions was correlated with decreased levels of FoxF1 mRNA. In situ
hybridization studies demonstrated that the defective primary lung-bud
development in early Foxf1(+/
) embryos was associated with
fewer pulmonary mesenchymal-epithelial interfaces. Defects in branching
morphogenesis in the Foxf1(+/
) embryos were associated
with altered expression of the fibroblast growth factor-10, bone
morphogenetic protein-4, and the Gli3 transcription factor, which are
known to influence primary lung-bud development.
fibroblast growth factor-10; Gli3; bone morphogenetic protein-4; Patched; winged-helix transcription factor; defective lung-bud formation
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INTRODUCTION |
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FORMATION OF THE MOUSE LUNG begins at 9.5 days postcoitum (pc) as a ventral outpouching of the foregut endoderm that invades the adjacent mesenchyme. Reciprocal interactions between the splanchnic mesenchyme and the respiratory epithelium support dichotomous branching that forms the left and right primary bronchi (10, 27). By 11 days pc, four right and one left primary lung bronchi are formed that define the lung lobes. Subsequently the conducting airways are formed by the process of branching morphogenesis (10, 27). Extensive branching of the lung tubules and thinning of the mesenchyme occur during the canalicular stage of lung development (16.5-17.5 days pc), which results in formation of prealveolar saccules lined by epithelial cells that are closely juxtaposed with the mesodermally derived pulmonary vasculature and stroma. In the terminal sac stage (17.5 days pc to postnatal day 5) of lung development, lung surface area increases by the septation of prealveolar saccules to form alveoli. The mature alveoli are lined by specialized pulmonary epithelial cells (type I and type II cells) and are vascularized by an extensive capillary bed that facilitates gas exchange.
A number of polypeptide factors have been implicated in lung-bud
formation and branching morphogenesis (reviewed in Refs. 10, 27) including Sonic hedgehog (Shh),
keratinocyte growth factor (KGF), bone morphogenetic protein-4 (BMP-4),
hepatocyte growth factor (HGF), fibroblast growth factor (FGF)-10, and
the FGF receptors. The FGF-10 signal transduction pathway appears to
play a critical role in lung-bud formation and branching morphogenesis. Fgf10(/
) embryos form no bronchial buds (10, 26,
28). Expression of Fgf10 occurs at the onset of lung
development in the splanchnic mesoderm surrounding the lung buds, and
FGF-10 serves as a chemoattractant during lung-bud formation (3,
10) and enhances proliferation of endodermally derived cells in
vitro (2, 17). Likewise, the pulmonary epithelial
transcription factors thyroid transcription factor-1 (TTF-1), GATA-6,
hepatocyte nuclear factor (HNF)-3
[also known as forkhead box
(Fox)a2], and HNF-3/forkhead homolog (HFH)-4 (also known as FoxJ1)
have been implicated in lung-branching morphogenesis, cell
differentiation, and lung-selective gene expression (6,
23).
The mammalian Fox genes are an extensive family of transcription
factors (13) that share homology in the winged-helix DNA binding domain (4). Fox family members regulate
transcription of genes critical for embryonic development, cellular
proliferation, and metabolism (Reviewed in Refs. 6,
7, 12, 16, 23, 30). One of these family members, Foxf1 [also
known as Hfh8 or forkhead-related activator
(Freac)-1], is first expressed during gastrulation in a
subset of mesodermal cells arising from the primitive streak region
that contributes to the extraembryonic and lateral mesoderm
(24). Consistent with this early expression pattern,
Foxf1(/
) embryos die in utero from defects in
extraembryonic and lateral mesoderm differentiation (19).
During organogenesis, high levels of FoxF1 expression persist in the
mesenchyme of the respiratory and gastrointestinal tracts. FoxF1 RNA is
expressed at mesenchymal-epithelial interfaces involved in lung and gut morphogenesis (20, 24). In the adult mouse, FoxF1 RNA is
detected in smooth muscle layers of pulmonary bronchioles, lamina
propria of the stomach and intestine, and alveolar endothelial cells
(15, 24).
Targeted ablation of the Foxf1 gene caused early embryonic
lethality (19), whereas severe pulmonary abnormalities
were observed in heterozygous Foxf1 mutant mice (14,
18). The winged-helix DNA binding domain of Foxf1 was
replaced by an in-frame insertion of a nuclear localizing
-galactosidase (
-gal) gene, which disrupted the function of the
mouse gene in vivo (14). Approximately 55% of newborn
Foxf1(+/
) heterozygous mice died of severe lung
hemorrhage. The severity of pulmonary abnormalities in the
Foxf1(+/
) mice correlated with diminished levels of FoxF1
mRNA [designated as low-Foxf1(+/
) mice]. Defects in lung
alveolarization and development of the alveolar capillaries were
observed in the low-Foxf1(+/
) newborn mice. Lung
hemorrhage in severely affected Foxf1(+/
) mice was
coincident with disruption of the mesenchymal-epithelial cell
interfaces in the alveolar and bronchiolar regions of the newborn lung
and was associated with increased apoptosis and reduced surfactant protein B (SP-B) expression. Moreover, the lung defect associated with the Foxf1(+/
) mutation was accompanied by
reduced expression of vascular endothelial growth factor (VEGF)
receptor-2 [fetal liver kinase-1 (Flk-1)], BMP-4, as well as the lung
Kruppel-like factor and T-box (Tbx2-Tbx5) transcription factors.
The mechanisms by which FoxF1 mediates changes in lung morphogenesis
remain unclear; however, recent studies demonstrated that
Foxf1(+/) lungs exhibit lobe fusions and that pulmonary expression of FoxF1 was undetectable in Shh(
/
) mouse
embryos, which suggests that Shh signaling is essential for
FoxF1 expression (18). However, analysis of pulmonary
genes with expression that was altered and associated with the lobular
defect in Foxf1(+/
) embryonic lungs remains
uncharacterized. The present study was conducted to determine the early
developmental defects leading to lung fusions in the
Foxf1(+/
) embryos and to identify potential genetic
pathways influenced by diminished pulmonary levels of FoxF1. In situ
hybridization of mesenchymal and epithelial marker genes [e.g.,
Foxf1, Patched (Ptc), Shh]
revealed that as early as 11 days pc, defects were observed
in Foxf1(+/
) embryonic lungs that were associated with
reduced expression of FGF-10, BMP-4, and the Gli3 transcription factor.
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MATERIALS AND METHODS |
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Heterozygous Foxf1 mice.
The generation of Foxf1(+/) mice was described previously
(14). Because the nuclear-localizing
-gal gene is
expressed at the targeted locus under control of FoxF1 regulatory
sequences,
-gal enzyme staining was used for analysis of FoxF1
expression. Heterozygous male mice were originally kept in a 129/Black
Swiss mouse background. For generation of the heterozygous embryos for morphological and RNA analysis, male breeder mice were mated with wild-type (wt) CD-1 female mice in the evening, and vaginal plugs were
checked in the morning. Noon of the day of appearance of a vaginal plug
was designated as 0.5 days pc. Dams were killed by carbon dioxide
asphyxiation at various days of gestation, and the embryos were removed
by caesarian section. Tail samples were used for genotyping by PCR
analysis as described previously (14).
-Gal staining.
Staining for
-gal was performed according to Clevidence and
colleagues (5) with a few modifications. Briefly, embryos and dissected lungs were fixed for either 30 min (11-14 days pc) or 1 h (older than 14 days pc) in 2% formaldehyde-0.2%
glutaraldehyde [with 0.02% Nonidet P-40 (NP-40) and 0.01% sodium
deoxycholate] in PBS at pH 7.8. Color development was done at 30°C
in the presence of 5 mM potassium ferrocyanide, 5 mM ferricyanide, 2 mM
magnesium chloride, 0.02% NP-40, 0.01% sodium deoxycholate, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-
-D-galactoside (X-Gal)
as described previously (5). After staining was completed,
samples were washed with PBS and postfixed in 4% paraformaldehyde
(PFA) overnight at 4°C, paraffin embedded, and sectioned as described
previously (14). For analysis of embryonic lung
morphology, dissected lungs were fixed overnight in 4% PFA at 4°C
and subsequently washed three times in PBS. Lungs were photographed
using a Nikon dissecting microscope and the MDS-120 digital microscopy
system (Kodak).
In situ hybridization with digoxigenin-labeled probes. Whole-mount lung in situ hybridization was carried out according to published protocols (9). Except for the FoxF1 (HFH-8) cDNA (24), the DNA clones used for generation of digoxigenin-labeled RNA probes were generated by RT-PCR of total RNA isolated from 9.5-day pc embryos or from adult mouse lungs. RT-PCR was performed with a SuperScript One-Step RT-PCR kit (GIBCO-BRL) according to the manufacturer's instructions. The recovered DNA fragments were subcloned in pBluescript SK (Stratagene), and the sequence of the clones was confirmed by dideoxy sequencing. T3 or T7 RNA polymerase in the presence of digoxigenin-labeled UTP generated sense and antisense riboprobes from linearized plasmid DNA by restriction-enzyme digestion and in vitro transcription. The relevant information regarding the DNA clones generated is listed in the following order: name of the gene, GenBank accession number, length of fragment generated, and DNA sequence amplified: Ptc, NM008957, 419 bp fragment, 3,450-3,954; Fgf10, NM008002, 480 bp, 80-609; VegfA, M95200, 481 bp, 497-977; Bmp4, D14814, 318 bp, 7,101-7,127 and 8,131-8,423; Shh, X76290, 311 bp, 516-826; Gli1, AB025922, 529 bp, 2,934-3,462; Gli2, X99104, 453 bp, 1,040-1,502; and Gli3, NM008130, 516 bp, 1,044-1,560.
RNA extraction and RNase protection assay.
Mouse lungs were dissected from 14-day-pc wt and Foxf1(+/)
embryos, and total lung RNA was prepared by an acid
guanidium-thiocyanate-phenol-chloroform extraction method using RNA
STAT-60 (Tel-Test B, Friendswood, TX). The RNase protection assay was
performed with [32P]UTP-labeled antisense RNA synthesized
from plasmid templates with the appropriate RNA polymerase as
previously described (25). RNA probe hybridization, RNase
ONE (Promega, Madison, WI) digestion, electrophoresis of RNA-protected
fragments, and autoradiography were performed as described previously
(15, 25). Cyclophilin probes were added to the same
hybridization reaction for normalization of RNA among lung RNA samples.
Quantitation was performed on scanned X-ray films using BioMax 1D
image-analysis software (Kodak). The mouse FoxF1 RNase protection probe
(Foxf1 cDNA nucleotides 437-816) was generated from
Foxf1 cDNA sequences located outside of the FoxF1/
-gal
gene-targeting vector (14, 15) and hybridizes selectively
to mRNA transcribed from the wt Foxf1 locus.
Foxf1(+/
) 14-day-pc embryos were divided on the basis of
pulmonary levels of FoxF1 mRNA using RNA protection assays: either
high-Foxf1 levels (~50% of Foxf1 levels in wt
lungs) or low-Foxf1 levels (20% of the Foxf1
levels in wt lungs). The low-Foxf1(+/
) mouse lung was associated with lung hemorrhage and perinatal lethality
(14). RNase protection probes for Flk-1 and BMP-4 were
described previously (14) and probes for the Gli2 and Gli3
transcription factors were described earlier.
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RESULTS |
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-Gal staining reveals mesenchymal expression in developing
Foxf1(+/
) lung.
In 9.5-day-pc Foxf1(+/
) embryos,
-gal staining derived
from the Foxf1 gene promoter was observed in the mesenchyme
of the foregut region containing the respiratory diverticulum that
gives rise to both the upper and lower respiratory regions (Fig.
1A). High levels of
-gal
staining persisted in the mesenchyme of the developing lung (10.5 days
pc), oral cavity, intestine, and allantois (Fig. 1, B and
D). In 12-day-pc Foxf1(+/
) embryos, high levels of
-gal staining were observed in the mesenchymal cells of the oral
cavity, lung, stomach, intestine, and intersomitic arteries and
in mesenchymal cells throughout the conducting airways of the lung
(Fig. 1, C and E). More intense
-gal
staining was evident in the mesenchyme juxtaposed to the bronchiolar
epithelial cells (12 days pc; Fig. 1E), the former giving
rise to the bronchiolar smooth muscle layer. These expression studies
reveal that FoxF1 is expressed in lung mesenchyme at sites
of interaction with epithelium.
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Defect in primary lung-bud formation in
Foxf1(+/) embryonic lungs.
By 11 days pc, the lung consists of five distinct lung buds, which form
the five lobes that are characteristic of the adult mouse lung (Fig.
2, A and D). The
formation of the four right lung buds occurs in the following
sequential order: dorsal cranial, lateral middle, caudal, and ventral
accessory bud (reviewed in Ref. 10). Furthermore,
the right and left lungs possess several lateral buds that undergo
branching morphogenesis but fail to develop into discrete lobes.
As early as 10.5-11 days pc, the Foxf1(+/
) lungs
displayed altered lung-bud orientation and diminished branching
morphogenesis (Fig. 2, B, C, E, and F). These
Foxf1(+/
) lung defects commonly included a caudal and
lateral shift in the orientation of the accessory lobe (Fig. 2,
arrowhead) and a lateral shift in the middle (Fig. 2, B, C,
E, and F) and cranial lobe positions (data not shown).
These studies indicate that FoxF1 is required for location site and
extent of branching morphogenesis of the lung buds.
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Right-lobe fusions of the embryonic Foxf1(+/)
lung.
As a consequence of the early defect in lung-bud formation, pairwise
ventral and dorsal views of either 12-day-pc (Fig.
3, C-F) and 13-day-pc
(Fig. 3, G and H) Foxf1(+/
) lungs or
12-day-pc wt lungs (Fig. 3, A and B) revealed a
range of fusion defects in the right Foxf1(+/
) pulmonary
lobes. Approximately 90% of Foxf1(+/
) lungs displayed a
fusion of the accessory lobe with the caudal lobe (Fig. 3,
E-H) that resulted from a range of altered positions of
the accessory lobe. Although this lobe-fusion defect was absent in 10%
of Foxf1(+/
) lungs, the orientation of the accessory lobe
was shifted caudally (Fig. 3, C and D).
Furthermore, embryonic Foxf1(+/
) lungs displayed fusion of
the right cranial and middle lobes, which was likely caused by a
lateral shift in the orientation during lung development (Fig. 3,
arrows). In severe forms, the Foxf1(+/
) lung-fusion
defect was associated with an angular shift of the cranial and middle
lobes, which merges them in the same plane [Fig.
4D; 18-day-pc
Foxf1(+/
) lung]. Cross sections of 14.5-day-pc wt
embryonic lung demonstrated distinct separation of the five lung lobes
and bronchi (see Fig. 3I). In contrast, examination of a
severely fused Foxf1(+/
) embryonic lung at the same
thoracic level revealed fusion of the cranial, middle, and caudal lobes
associated with aberrantly juxtaposed secondary bronchi (see Fig.
3J).
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Fewer mesenchymal-epithelial interfaces in
Foxf1(+/) lungs.
To examine the expression pattern of signaling molecules in the
Foxf1(+/
) lungs, in situ hybridization analysis was
performed on 11-day-pc lungs. Whole-mount in situ hybridization for
FoxF1 and the putative Shh receptor Ptc were compared with 11-day-pc wt
and Foxf1(+/
) heterozygous type (ht) lungs. FoxF1 and Ptc mRNA expression overlapped in the distal mesenchymal cells of the
developing wt lung buds (Fig. 6,
A and B, arrowheads). The defective lung-bud
formation in Foxf1(+/
) mice was associated with fewer
mesenchymal regions that expressed the Foxf1 and
Ptc genes (Fig. 6, A and B). Likewise,
embryonic Foxf1(+/
) lungs developed fewer distal bronchial
epithelial regions expressing Shh and VEGFA (1, 22)
compared with wt lungs (Fig. 6, C and D).
|
Diminished distal mesenchymal expression of FGF-10 in
10- to 11-day-pc Foxf1(+/) lungs.
Whole-mount in situ hybridization studies revealed that
Foxf1(+/
) lungs exhibited diminished FGF-10 hybridization
signals in the ventral view, which allowed visualization of the right, middle, cranial, and accessory lung buds (Fig. 6E). In
contrast, FGF-10 RNA was unaltered in the Foxf1(+/
) distal
lung mesenchyme in a dorsal view, which visualizes the left and caudal
lung buds (Fig. 6F). Likewise, at 10 days pc,
Foxf1(+/
) lungs exhibited diminished FGF-10 RNA in the
distal mesenchyme in the ventral view (Fig. 6G) but not in
the dorsal view (Fig. 6H). In light of the critical role of
FGF-10 in lung-bud formation and branching morphogenesis, it is likely
that reduced mesenchymal expression of FGF-10 in the developing
Foxf1(+/
) lungs contributes to the defective bud formation
of the right ht lung.
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Defective orientation of vessels in Foxf1(+/)
embryos.
Fusion of the Foxf1(+/
) embryonic lung lobes was
associated with fusion of correspondingly large pulmonary blood
vessels. The invariant relationships among the major veins (white) and arteries (black) and the bronchial airway (red) are schematically shown
in Fig. 9D.
Foxf1(+/
) embryonic lungs (19 days pc) with mild fusion of
the accessory and caudal lobes (Fig. 9B) showed misplacement
and fusion of vessels. For example, instead of the middle lobe vein
entering the ventral face of the middle lobe (Fig. 9A), it
entered the cranial side of the middle lobe in Foxf1(+/
) embryonic lungs (Fig. 9B). Furthermore, even
Foxf1(+/
) lungs with mild phenotype displayed fusion of
the caudal and accessory lobe veins within the abnormally fused lung
lobes (Fig. 9E). Foxf1(+/
) embryos with severe
fusions of the right lung lobes also possessed complete fusion of the
accessory and caudal pulmonary veins, which bifurcated within the fused
lobe (schematically shown in Fig. 9F). In addition, small
abnormal veins were found that connected the cranial and middle lobe
veins (schematically shown in Fig. 9F, shared veins). In
more severely affected Foxf1(+/
) lungs, multiple vein
fusions were observed between the cranial and middle lobe veins as well
as between the caudal and accessory veins (Fig. 9C and
schematically shown in Fig. 9G). These studies
show that fusion of the Foxf1(+/
) lung lobes also resulted
in fusion of the major pulmonary veins within these lobes.
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DISCUSSION |
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In a previous study (14), we reported that 55% of
the newborn Foxf1(+/) mice died of severe lung hemorrhage
and that the severity of the pulmonary abnormalities correlated
with significantly diminished FoxF1 mRNA [low
Foxf1(+/
)]. Defects in lung alveolarization and
vasculogenesis were observed in low-Foxf1(+/
)
newborn mice and were associated with reduced expression of Flk-1 and
BMP-4 (14). Pulmonary hemorrhage was not observed in a
subset of newborn Foxf1(+/
) mice expressing wt levels of
FoxF1 mRNA [designated as high-Foxf1(+/
) mice], and
these ht mice exhibited normal life spans. In this study, we show that
Foxf1(+/
) embryos exhibited defects in lung-bud formation
and branching that lead to fusion of the right lung lobes and cause
secondary fusion anomalies of pulmonary veins. Our studies indicate
that defective branching morphogenesis changes the orientation of the
right lung lobes in Foxf1(+/
) lungs in a process that
produces either mild or severe fusion of right lung lobes in ht
embryos. Fusion of the cranial and middle lobes and among the cranial,
middle, and caudal lobes in the lung were observed in severely affected
embryos, which displayed 20% of FoxF1 levels found in wt embryonic
lungs. The severe Foxf1(+/
) lung-fusion defect accounted
for the majority of newborn mice that died perinatally, and all of them
were shown to express reduced pulmonary levels of FoxF1
(14). The most prevalent Foxf1(+/
) lung
defect consisted of a mild fusion between the caudal and accessory
right lobes resulting in a lateral shift in the position of the
accessory lobe. This mild lung-fusion defect was found in surviving
adult Foxf1(+/
) mice in which wt levels of FoxF1 mRNA were
observed in the lungs. Taken together, our results suggest that severe
fusion of the right lung lobes is associated with significant
reductions in pulmonary FoxF1 levels and suggest that its expression is
critical for proper lung morphogenesis.
Although a recent study reported that Foxf1(+/) CD-1 mouse
embryos exhibit esophageal atresia, tracheoesophageal fistula, skeletal
abnormalities, and lung-lobe fusions, the Foxf1(+/
) lungs
were not used to identify pulmonary genes with altered expression that
was associated with the lobular fusion (18). Our current study carried out a developmental expression analysis of
Foxf1(+/
) mouse lungs to identify genes with altered
expression due to FoxF1 haploinsufficiency and that may contribute to
the lung-fusion phenotype. In situ hybridization studies with
mesenchymal (Foxf1, Ptc) and epithelial
(Shh, VegfA) marker genes revealed that as early
as 11 days pc, Foxf1(+/
) lungs exhibited fewer
mesenchymal-epithelial interfaces. We also found that severe lung-lobe
fusions in Foxf1(+/
) mice were associated with reduced
pulmonary expression of BMP-4, a signaling molecule that is essential
for proper lung morphogenesis (28) and may therefore
contribute to defective lobe formation in Foxf1(+/
) lungs.
Diminished pulmonary expression of FoxF1 reduced lung-bud formation and
branching morphogenesis in association with decreased mesenchymal
expression of FGF-10 in the periphery of right middle, cranial, and
accessory lobes at 10-11 days pc in Foxf1(+/
)
embryos. In 12-day-pc Foxf1(+/
) lungs, we detected increased mesenchymal expression of FGF-10 in the distal tip of the
accessory lobe bud; FGF-10 expression is delayed in the
Foxf1(+/
) lungs. The initial positioning of the lung buds
was defective in the Foxf1(+/
) lungs. Genetic and in vitro
studies have implicated a role for FGF-10 as a chemoattractant that
influences the location of lung-bud formation (3, 10, 17, 26,
28). It is therefore likely that the altered temporal expression
pattern of FGF-10 in early embryonic Foxf1(+/
) lungs
contributes to observed defects in branching and lobulation. Because
FoxF1 expression is found at the onset of splanchnic mesoderm formation
(24), our studies are also consistent with the possibility
that wt FoxF1 levels are necessary for normal temporal expression of
FGF-10 in the splanchnic mesenchyme. Alternatively, FGF-10 and FoxF1
may function in the regulatory loop that maintains their expression.
The zinc-finger Gli transcription factors are homologs of the
Drosophila segment polarity gene cubitus interruptus, and,
in response to Shh signaling through its putative receptor
Ptc, the Gli proteins are transcriptionally active because they are not proteolytically cleaved (11, 21). Defective lung-bud
formation in Foxf1(+/) embryos were associated with
changes in the expression of the Gli3 transcription factor, which has
been implicated in lung-lobe formation and morphogenesis (6,
29). RNase protection assays demonstrated that
low-Foxf1(+/
) lungs (14 days pc) displayed a 70%
reduction in Gli3 mRNA. Decreases in Gli3 mRNA seen in
Foxf1(+/
) lungs may play a role in the aberrant defects in
lung budding. Defects in the right medial, caudal, and accessory lung
lobes were observed in Gli3(
/
) mice (8).
Furthermore, Foxf1(+/
) lungs possess reduced Shh
signaling, because fewer Ptc-expressing mesenchymal cells were present
in Foxf1(+/
) lungs. Potentially, the diminished number of
mesenchymal cells expressing Ptc in Foxf1(+/
) embryonic
lungs may lead to reduced Shh signaling and cause proteolytic cleavage
of Gli proteins that in general act as transcriptional repressors of
Gli target genes (11, 21). Taken together, our results
suggest that the combination of diminished Ptc-mediated Shh signaling
and reduction in Gli3 transcription-factor expression may contribute to
the lung-lobe fusion defects in Foxf1(+/
) embryos.
We also observed that the pulmonary veins were misplaced in the
Foxf1(+/) lungs, and this defect was associated with the severity of the lung fusions. Our previous data (14)
demonstrated that the peripheral pulmonary vasculature of
Foxf1(+/
) mice was disrupted as assessed by platelet
endothelial cell adhesion molecule (PECAM)-1 staining; however, PECAM-1
staining was normal in the large vessels. The defect in peripheral
vasculature was associated with reduced expression of VEGFA and Flk-1
in the developing Foxf1(+/
) lungs. Whether the observed
defects in orientation and fusion of large pulmonary vessels in
Foxf1(+/
) mice was a direct consequence of the decreased
FoxF1 mRNA is not entirely clear. Because the formation of the
pulmonary vasculature is closely related to the growth of the
conducting airway bronchioles, we propose that the defects in large
vessels in the Foxf1(+/
) lungs were secondary to defects
in the initial bud-site specifications.
In summary, we have shown that Foxf1(+/) embryos exhibited
defects in lung-bud formation and branching, which caused fusion of
lung lobes and pulmonary veins. Foxf1(+/
) newborn mice
with severely fused lung lobes died perinatally, and the lung defects correlated with the diminished pulmonary levels of FoxF1 expression. Early embryonic Foxf1(+/
) lungs exhibited diminished
numbers of mesenchymal-epithelial interfaces that may disrupt formation of lung branch points. Expression of FGF-10, BMP-4, and the Gli3 transcription factor was decreased in lungs of Foxf1(+/
)
embryos and may play a role in the disruption of branching
morphogenesis that was seen in the haploinsufficient FoxF1 mice.
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ACKNOWLEDGEMENTS |
---|
The authors thank Brian Shin and Jean Clark for excellent technical assistance and thank Pradip Raychaudhuri for critical review of the manuscript.
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FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-62446 (to R. H. Costa), HL-56387 (to J. A. Whitsett), and HL-41496 (to J. A. Whitsett).
Address for reprint requests and other correspondence: R. H. Costa, Dept. of Molecular Genetics (M/C 669), Univ. of Illinois at Chicago, College of Medicine, 900 S. Ashland Ave., Rm. 2220 MBRB, Chicago, IL 60607-7170 (E-mail: Robcosta{at}uic.edu).
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.
First published December 14, 2001;10.1152/ajplung.00371.2001
Received 19 September 2001; accepted in final form 8 December 2001.
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