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First published online 11 April 2007
doi: 10.1242/dev.02846
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1 Cardiovascular Institute, University of Pennsylvania, 956 BRB II/III, 421
Curie Blvd., Philadelphia, PA 19104, USA.
2 Department of Molecular Genetics and the Institute for Cellular and Molecular
Biology, University of Texas at Austin, TX, USA.
3 Department of Cell and Developmental Biology, University of Pennsylvania, 956
BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104, USA.
* Author for correspondence (e-mail: emorrise{at}mail.med.upenn.edu)
Accepted 5 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Foxp1, Foxp2, Lung development, Esophagus, T1alpha (podoplanin), Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the lung, the surfactant protein genes have acted as surrogate readouts
for the important transcriptional mechanisms underlying lung development.
These studies have lead to the identification of several transcription factors
including Nkx2.1 (Titf1 - Mouse Genome Informatics), Gata6, Foxa1/2 and
C/EBP
as important regulators of lung endoderm differentiation and
development (reviewed by Cardoso and Lu,
2006
). Many of these factors are part of large families of
proteins, which act both individually and cooperatively to control gene
transcription in tissues where multiple family members are expressed.
Among the transcription factor families known to be crucial for lung
development, the Fox family is of particular importance. Fox proteins are
characterized by their highly homologous DNA-binding domain, which forms a
`winged-helix' motif (Kaestner et al.,
2000). Fox genes are important regulators of foregut development,
including tissues such as the liver, pancreas, intestinal epithelium and lung.
Fox factors are crucial activators of lung-specific gene expression and both
Foxa1 and Foxa2 are important for airway morphogenesis and epithelial
differentiation in the lung. Loss of Foxa2 expression results in distinct
defects in alveolarization, whereas Foxa1-null mice exhibit transient
defects in lung epithelial differentiation
(Besnard et al., 2005;
Wan et al., 2004). Loss of
both genes results in a severe disruption in branching morphogenesis with a
concurrent loss of epithelial differentiation
(Wan et al., 2005). Such
redundancy is likely to be important for large transcription factor families
such as the Fox family that are crucial for tissue-specific development.
We have previously identified a subfamily of Fox factors, Foxp1/2/4, that
are highly expressed in distinct patterns in the developing airway epithelium
(Lu et al., 2002).
Loss-of-function of each gene resulted in distinct and severe defects in
cardiovascular, neural and hematopoietic development
(Hu et al., 2006;
Li et al., 2004b;
Shu et al., 2005a;
Wang et al., 2004). However,
it remains unclear what role these factors play in lung development, as
Foxp1 and Foxp4 mutants exhibit normal lung specification
but do not survive past E13.5. We show that Foxp2-null mice exhibit
defects in postnatal alveolarization. Given their overlapping patterns of
expression, redundancy of Foxp2 and Foxp1 was addressed by generating
Foxp2-/-;Foxp1+/- mutants. In contrast to
Foxp2-null animals, these compound mutants die at birth due to
increased severity in airway morphogenesis and differentiation defects leading
to respiratory failure. Furthermore,
Foxp2-/-;Foxp1+/- mutants have severe defects
in esophageal development, indicating a broader role in regulation of anterior
foregut development. These data identify Foxp2 and Foxp1 as crucial regulators
of lung airway morphogenesis and differentiation as well as esophageal muscle
development, pointing to a complex interplay amongst Foxp factors in the
regulation of anterior foregut development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wang et al.,
2004). Mice were kept on a C57BL/6x129SVj mixed background.
Foxp1+/- and Foxp2+/- were
intercrossed to generate
Foxp2+/-;Foxp1+/-, which were further
crossed with Foxp2+/- to generate
Foxp2-/-;Foxp1+/- animals. For timed matings,
noon of the day that the vaginal plug was observed was considered E0.5.
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;
Yin et al., 2006). The
following antibodies and concentrations were used in the histological studies:
SP-C (Chemicon #07-647, 1:500), CC10 (Santa Cruz T-18, 1:50), SP-B (Chemicon
#AB3436, 1:250), T1alpha (Developmental Studies Hybridoma Bank, University of
Iowa, mAb 8.1.1, 1:100), phospho-histone H3 (Cell Signaling Technologies
#9701, 1:500), smooth muscle -actin (Sigma clone 1A4, 1:200), Foxp1
antisera (1:400), Foxp2 antisera (1:400), MyoD (Novacastra NCL-MyoD1, 1:20).
Complete details on all histological procedures can be found at the University
of Pennsylvania Cardiovascular Institute Histology Core web site
http://www.uphs.upenn.edu/mcrc/histology/histologyhome.html.
Lung morphometry and proliferation index
The mean linear intercept (MLI) on E18.5 and postnatal lung samples was
calculated as follows, based on previously published protocols
(Neptune et al., 2003;
Thurlbeck, 1967). Digital
images were captured at both 200x and 400x magnification.
Horizontal, vertical and diagonal grid lines were overlaid and used to count
the number of alveolar septa intersections. MLI was calculated as follows:
length of grid lines divided by the number of intersections with alveolar
septa. Data are from three samples of each indicated genotype. Data were
analyzed using Student's t-test and values considered significant if
P<0.05.
Lung-to-body weight ratios were based on four embryos of each genotype at each age tested. To determine changes in E14.5 and E18.5 airspace luminal area, ImageJ software was used to compare distal airspace area in arbitrary pixel units. This was performed on four sections from each of three different lung samples of the indicated genotypes. Data were analyzed using Student's t-test and values considered significant if P<0.05.
A proliferation index for both the epithelial and mesenchymal cells in the indicated wild-type and mutant lungs was generated by counting the percentage of Ki-67-positive cells in five fields of view from three different embryos. Epithelial cells were selected on the basis of their position lining distal airways, whereas mesenchymal cells were selected on the basis of not lining airways. Data were analyzed using Student's t-test.
Quantitative RT-PCR (Q-PCR) and chromatin immunoprecipitation (ChIP) assays
Total RNA was isolated using Trizol (Invitrogen) and Q-PCR was performed
using the oligonucleotides listed in Table
1 and an Applied Biosystems 7900HT system with Syber Green
reaction mixture as previously described
(Lepore et al., 2005).
Chromatin was prepared from E18.5 mouse lung tissue using the ChIP Kit
(Upstate Biotechnology). Lung tissue was minced, fixed with 1% formaldehyde
and chromatin sheared by sonication to an average length of 500-600 bp. The
Foxp1 and Foxp2 antibodies used for immunoprecipitation have been described
previously (Lu et al., 2002).
Reverse cross-linked immunoprecipitated chromatin was subjected to PCR using
the oligonucleotides listed in Table
1.
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). Expression
plasmids for Foxp1 and Foxp2 and the 1.3 kb rat T1alpha luciferase reporters
have been described previously (Ramirez et
al., 1997; Shu et al.,
2001). Luciferase assays were performed 48 hours after
transfection using the Dual Luciferase Kit (Promega). All data are the average
of three assays performed in triplicate±s.e.m. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
To directly compare Foxp1 and Foxp2 expression in the lung and esophagus,
immunohistochemistry was performed on E14.5 embryos. Foxp1 was found to be
expressed at high levels in distal lung epithelium and at lower levels in more
proximal bronchial epithelium (Fig.
1A). Foxp1 expression was also observed in the developing
mesenchyme, most likely in endothelial precursors as Foxp1 is expressed in the
developing vascular endothelium later in development
(Fig. 1A)
(Lu et al., 2002). In contrast
to Foxp1, Foxp2 expression was restricted to distal lung epithelia and was not
expressed in the developing mesenchyme
(Fig. 1B). In adult lungs,
Foxp1 was found to be expressed primarily in alveolar epithelial type 2
(AEC-2) cells located in the corners of alveoli, whereas Foxp2 expression was
observed in AEC-2 as well as alveolar epithelial type 1 (AEC-1) cells, which
contain a more flattened nucleus in the walls of the alveolus
(Fig. 1C,D). Both Foxp1 and
Foxp2 were expressed in the muscular component of the developing esophagus,
whereas only Foxp1 was expressed in the developing esophageal epithelium
(Fig. 1E,F). These data
demonstrate both extensive overlap and distinct differences in Foxp1 and Foxp2
expression during foregut development.
Given the high-level expression of Foxp2 in distal lung epithelium, we
sought to determine whether there were defects in lung morphogenesis or
maturation in Foxp2-null mice. We have previously demonstrated that
Foxp2-null mice die approximately three weeks postnatally
(Shu et al., 2005a), a time
when active lung alveolarization occurs. Histological analysis revealed that
at E18.5, Foxp2-null lungs are morphologically similar to wild-type
littermates (Fig. 2A,D).
However, by P8, airways in Foxp2-null lungs appeared severely dilated
(Fig. 2B,E). This airway
dilation was also evident at P20 (Fig.
2C,F). Mean linear intercept analysis revealed significant
dilation of distal airspaces at P8 and P20, indicating postnatal
alveolarization defects in Foxp2-null mice
(Fig. 2G). Transmission
electron microscopy (TEM) was performed to visualize the alveolar airspaces at
high magnification. Little difference was observed in the number or morphology
of AEC-1 and AEC-2 cells (Fig.
2H,I).
Immunohistochemistry was performed on wild-type and Foxp2-null
mice to determine the effect that loss of Foxp2 expression had on lung
epithelial cell lineage differentiation. Expression of the AEC-2 cell markers
SP-B and SP-C and the Clara cell marker CC10 (also known as Sftpb, Sftpc and
Scgb1a1, respectively - Mouse Genome Informatics) were unaffected by loss of
Foxp2 (Fig. 3A,B,E-H). However,
expression of the AEC-1 cell marker T1alpha (also known as podoplanin - Mouse
Genome Informatics) was markedly increased in Foxp2-null animals
(Fig. 3C,D). Q-PCR also
revealed an increase in T1alpha expression, whereas expression of aquaporin 5,
another AEC-1-restricted marker gene, as well as other AEC-2 marker genes,
were unaffected (Fig. 3N).
Foxp2 and T1alpha proteins are co-expressed in the same cells in the distal
airways at E18.5, indicating that Foxp2 could have a direct affect on T1alpha
gene expression (Fig. 3I-K). In
addition to AEC-1 cells, T1alpha is also expressed in the lymphatic
endothelium of the lung (Schacht et al.,
2003). However, T1alpha expression in this cell type did not
appear to be affected by loss of Foxp2
(Fig. 3L,M). Given the expanded
nature of the airways, these data suggest either disruption in alveolar
epithelial differentiation or increased numbers of AEC-1 cells in the distal
airways of Foxp2-null mice. Since TEM studies did not indicate an
increased number of AEC-1 cells in Foxp2-/- mutants, these
data suggest that the increase in T1alpha is due to Foxp2 acting directly on
T1alpha gene expression. Thus, Foxp2 is required for postnatal lung
alveolarization and regulation of the AEC-1 cell-restricted gene T1alpha.
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; Shu et al.,
2001). Three consensus Fox DNA-binding sites are located in the
1.3 kb rat T1alpha promoter, two of which are conserved in the mouse gene
(Fig. 3O)
(Ramirez et al., 1997). This
promoter has been used to direct lung epithelial gene expression in transgenic
mice in previous studies (Ramirez et al.,
1999). To determine whether Foxp2 could repress the T1alpha
promoter, a luciferase reporter plasmid with the 1.3 kb rat T1alpha promoter
was co-transfected into NIH-3T3 cells along with expression plasmids for Foxp2
and Foxp1. Expression of either Foxp2 or Foxp1 repressed the T1alpha promoter
approximately fivefold (Fig.
3O).
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Loss of a single Foxp1 allele in a Foxp2-null background leads to dramatic embryonic lung defects
The above studies suggested that Foxp1 acts cooperatively with Foxp2 to
regulate lung epithelial gene expression. First, Foxp1 and Foxp2 are
co-expressed at high levels in distal lung epithelium during development.
Second, both Foxp1 and Foxp2 are found associated with conserved Fox
DNA-binding sites on the T1alpha promoter in vivo. Third, the T1alpha promoter
was repressed by both Foxp1 and Foxp2. Finally, there is no compensatory
upregulation of Foxp1 or Foxp4 in Foxp2-null lungs (see Fig. S1 in
the supplementary material). We generated
Foxp1-/-;Foxp2-/- mutants, but they die prior
to E11.5 and are severely runted, not allowing an accurate assessment of lung
development (data not shown). Thus, we generated
Foxp2-/-;Foxp1+/- mutants to determine whether
loss of a single Foxp1 allele in addition to complete loss of Foxp2
expression would lead to increased severity in lung defects.
In contrast to Foxp2-null mice, Foxp2-/-;Foxp1+/- mice did not survive beyond the neonatal stage (Table 2). However, at least some Foxp2-/-;Foxp1+/- animals did survive gestation, although the exact percentage is difficult to determine owing to the fact that Foxp1 and Foxp2 are linked on mouse chromosome 6 (Table 2). This suggested that loss of a single Foxp1 allele in a Foxp2-null background increased the severity of defects in specific tissues where they are co-expressed during development, such as the lung. Histological analysis revealed severe lung airway defects as early as E14.5 (Fig. 4A-J). These included dilated airways and reduced lung size, indicating defects in branching morphogenesis and cellular proliferation in the lung. Lung-to-body weight ratios were significantly reduced for Foxp2-/-;Foxp1+/- mutants at both E17.5 and P0 (Table 3). Measurement of distal airspace luminal area reveal an almost threefold increase at E14.5 and an almost twofold increase at E18.5, further indicating defects in lung airway morphogenesis (Fig. 4K).
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Given the defects observed in
Foxp2-/-;Foxp1+/- lungs, we performed in situ
hybridization and immunohistochemistry for expression of genes that function
in airway epithelial development and alveolarization to determine the extent
of airway epithelial differentiation in these embryos. As observed at E18.5,
expression of the gene encoding SP-C was not disrupted at E16.5, although
airway dilation was evident (Fig.
5A,B). Expression of the genes encoding Nkx2.1, sonic hedgehog
(Shh), SP-B, Foxa2 and Gata6 were unchanged in
Foxp2-/-;Foxp1+/- lungs
(Fig. 5C-L). However,
expression of the genes encoding N-myc (Mycn - Mouse Genome Informatics) and
Hop (Hod - Mouse Genome Informatics) were significantly reduced as assessed by
in situ hybridization in Foxp2-/-;Foxp1+/-
lungs (Fig. 5M-P). This
reduction was confirmed by Q-PCR in wild-type and
Foxp2-/-;Foxp1+/- lungs at E16.5
(Fig. 5Q). N-myc is a crucial
regulator of distal lung development and its loss leads to dramatic defects in
airway morphogenesis, including defects in airway epithelial proliferation
(Okubo et al., 2005). Hop is a
homeodomain protein expressed in the developing airway epithelium in a unique
temporal expression pattern and loss of Hop leads to lung alveolarization
defects and partial perinatal lethality
(Yin et al., 2006). Thus, the
reduction in N-myc and Hop expression, but unchanged expression of other lung
epithelial markers, suggests that Foxp2 and Foxp1 cooperatively regulate a
specific transcriptional program required post-specification to regulate lung
epithelial differentiation and airway morphogenesis. The significant loss in
N-myc expression could be responsible for the observed defects in airway
morphogenesis as a complete loss in N-myc results in dramatic loss in
branching morphogenesis (Okubo et al.,
2005).
Given the relatively small size of
Foxp2-/-;Foxp1+/- lungs and the loss in N-myc
expression, cell proliferation and apoptosis was assessed in wild-type and
mutant lungs. Immunohistochemistry using the cell proliferative marker Ki-67
(Mki67 - Mouse Genome Informatics) revealed a significant reduction in cell
proliferation in the epithelia and mesenchyme of
Foxp2-/-;Foxp1+/- lungs
(Fig. 6A-C). However, TUNEL
staining did not reveal any significant changes in apoptosis in
Foxp2-/-;Foxp1+/- lungs (data not shown). These
data suggest that the reduced cell proliferation may account for the reduced
lung size in Foxp2-/-;Foxp1+/- mutants. To
determine the underlying mechanism by which Foxp1 and Foxp2 regulate cell
proliferation in the lung, expression of cyclin D1 and the cyclin-dependent
kinase inhibitors (CDKI) p21, p27 and p57 (Cdkn1a, Cdkn1b and Cdkn1c,
respectively - Mouse Genome Informatics) were assessed. Remarkably, we
observed reduced levels of cyclin D1 and increased levels of p57 in the
airways of Foxp2-/-;Foxp1+/- mutants
(Fig. 6D-G). Changes in
expression of p21 and p27 were not observed (data not shown). These data,
along with previous studies showing that loss of Foxp1 in the heart leads to
increased p21 and decreased p27 (Wang et
al., 2004), indicate that Foxp1 and Foxp2 regulate cell cycle
regulators in a cell type-specific manner. Together with the dramatic loss in
N-myc and Hop expression, these data suggest that Foxp1 and Foxp2
cooperatively regulate cell proliferation programs in lung epithelia required
for proper growth of the airways.
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-actin (sm-actin) was used to delineate the muscular layers within the
developing esophagus. In wild-type mice, sm-actin expression was observed in
three layers of the developing esophagus: an inner submucosal layer directly
adjacent to the epithelium and two outer layers representing the
circumferential and longitudinal layers
(Fig. 7G,H). In contrast to
wild-type embryos, Foxp2-/-;Foxp1+/- mutants
contained only a single outer layer of muscle in addition to the submucosal
layer adjacent to the esophageal endoderm
(Fig. 7I,J).
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;
Rishniw et al., 2003). To
determine whether there was a normal contribution of skeletal muscle to
Foxp2-/-;Foxp1+/- esophagus, tissues were
stained for the presence of skeletal muscle development using an antibody to
the skeletal muscle-specific transcription factor MyoD (MyoD1 - Mouse Genome
Informatics). As expected, wild-type esophagus at E18.5 contained numerous
MyoD-positive cells in the outer muscular layer
(Fig. 7K). By contrast,
Foxp2-/-;Foxp1+/- mutants had no detectable
MyoD-positive cells in the muscle surrounding the esophagus
(Fig. 7L).
The loss of skeletal and smooth muscle development in the esophagus of
Foxp2-/-;Foxp1+/- mutants could be due to a
loss of proliferation of these cells, their apoptosis during development, or
to defects in differentiation of these cell types. Therefore, we assessed cell
proliferation and apoptosis in wild-type and
Foxp2-/-;Foxp1+/- mutant esophagi. No changes
in either proliferation as assessed by Ki-67 immunostaining, or in apoptosis
as assessed by TUNEL staining, were observed
(Fig. 8). These data suggest a
loss of esophageal muscle differentiation in
Foxp2-/-;Foxp1+/- mutants. However, given that
a complete loss of skeletal muscle in the esophagus did not result in a
phenotype as dramatic as that exhibited by
Foxp2-/-;Foxp1+/- mutants
(Kablar et al., 2000), the loss
of skeletal muscle in these mutants does not completely account for the severe
reduction in muscle mass or dilated appearance of the esophagus. Together,
these results indicate that both smooth and skeletal muscle differentiation is
disrupted in the esophagi of Foxp2-/-;Foxp1+/-
mutants.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Li et al., 2004b;
Shu et al., 2005a;
Wang et al., 2004). In this
report, we show that Foxp2 and Foxp1 work cooperatively to regulate lung gene
expression, airway morphogenesis and esophageal muscle development. Given that
both factors are highly expressed in the developing airway epithelia and
esophageal muscle, these data demonstrate that Foxp1 and Foxp2 act in an
allelic dose-dependent manner to regulate gene expression in these cell
types.
Since Foxp1/2 are known transcriptional repressors
(Li et al., 2004a), the
increased expression of T1alpha in Foxp mutants is provocative and is likely
to reflect these factors acting directly on the T1alpha promoter to restrict
its expression in alveolar epithelial cells. Our data showing in vivo
occupancy of the T1alpha promoter by Foxp1 and Foxp2 support this concept.
T1alpha expression is restricted to AEC-1 cells in late gestation, but it is
expressed throughout distal airway epithelium during early lung development
(Ramirez et al., 1999;
Ramirez et al., 1997). T1alpha
is also expressed in lymphatic endothelium in the lung and elsewhere
(Schacht et al., 2003).
Interestingly, we did not observe an increase in T1alpha expression in the
lymphatic endothelium in the lung or elsewhere in the developing embryo. This
may be explained by the fact that Foxp2 is not expressed in lymphatic
endothelium (data not shown).
The function of T1alpha is largely unknown, but T1alpha-null mice exhibit
both airway epithelial defects and lymphatic endothelial defects
(Ramirez et al., 2003;
Schacht et al., 2003). Based
on the protein structure, T1alpha is a mucin-type glycoprotein with extensive
O-glycosylation (Kato et al.,
2003). T1alpha-null mice have defects in lymphatic vascular
patterning leading to large dilated lymphatics
(Schacht et al., 2003).
T1alpha also appears to regulate lymphatic endothelial cell migration and
adhesion. Overexpression of T1alpha in microvascular endothelial cells leads
to increased cell migration and adhesion
(Schacht et al., 2003). AEC-1
cells cover the vast majority of alveolar airspace in the late gestational and
postnatal lung. Undoubtedly, cell migration and adhesion play a key role in
the ability of AEC-1 cells to form the thin gas-permeable interface by
spreading their cytoplasmic processes to cover such a vast surface area. Given
its potential affects in regulating both lung epithelial and lymphatic
endothelial differentiation, increased expression of T1alpha could lead to
defects in late-stage AEC-1 cell differentiation, which in turn leads to
defective lung alveolarization, a developmental process whereby AEC-1 cells
help to remodel the distal airspaces to produce the well established alveolus
required for efficient gas exchange in the lung. In support of this concept,
gain-of-function experiments in which T1alpha is overexpressed in distal lung
epithelium using the human SP-C (SFTPC - Human Gene
Nomenclature Database) promoter, showed that increased T1alpha expression
leads to increased postnatal mortality after hyperoxic injury
(Girod et al., 1999). Although
the data presented in this study are preliminary and do not suggest a distinct
mechanism to explain these results, they do suggest that increased levels of
T1alpha might predispose the lung to additional defects or injury leading to
alveolar dysfunction. Gain-of-function experiments of crucial signaling and
transcriptional regulators in the lung, including Shh, Bmp4 and Gata6,
revealed severe defects in late airway epithelial development, including
alveolarization defects (Bellusci et al.,
1997; Liu et al.,
2003; Weaver et al.,
1999). These studies support the concept that transcriptional
repression to control temporal and spatial gene expression is an important
mechanism for regulating lung epithelial morphogenesis and differentiation.
Thus, increased expression of T1alpha caused by the loss of Foxp2-mediated
repression could contribute to the lung defects in Foxp2-null
animals, but is unlikely to be the sole cause. Given the dearth of information
regarding the direct targets of Foxp1/2 and the function of T1alpha in vivo,
these data add critical insight into how these factors regulate lung
epithelial differentiation.
Previous studies have implicated Foxp1 as a tumor suppressor gene,
with reduced expression observed in cancers from several tissues including the
lung. Foxp1 maps to chromosome 3p14.1, a region commonly associated
with loss of heterozygosity in several forms of cancer
(Banham et al., 2001). In the
lung, Foxp1 expression is reduced in lung tumors induced with the carcinogenic
reagent N-nitrosobis(2-hydroxypropyl)amine
(Shimizu et al., 2006).
Moreover, loss of Foxp1 leads to an aberrant increase in cardiomyocyte
proliferation and defective differentiation
(Wang et al., 2004). Less is
known about the potential oncogenic role of Foxp2 and Foxp4. In contrast to
the lung and heart, esophageal smooth muscle proliferation does not appear to
be affected in Foxp2-/-;Foxp1+/- mutants. This
could be explained by an inability to detect a short temporal window of
decreased proliferation with the techniques used, or to intrinsic differences
in the roles of Foxp1 and Foxp2 in esophageal smooth muscle. The decreased
proliferation in both the epithelial and mesenchymal compartments of
Foxp2-/-;Foxp1+/- lungs is somewhat
counterintuitive to the result observed in lung tumors and cardiomyocytes.
However, the significant decrease in N-myc, which plays a crucial role in lung
epithelial cell proliferation, may override any increase in proliferation from
loss of Foxp1 and Foxp2 expression. In the adult lung after injury, N-myc
expression could be reactivated to help in re-epithelialization of the
airways, and in this instance Foxp1 (or Foxp2) may be required for its proper
expression. Loss or gain of Foxp1/2 expression could lead to an aberrant
injury response in the lung, leading to epithelial hyperplasia and eventually
tumorigenesis. Future studies to specifically delete Foxp1 in the
postnatal lung will be required to determine whether this gene acts as a tumor
suppressor in the lung.
Most Fox factors exist in small subfamilies of highly related factors that
have overlapping patterns of expression. Foxp1/2/4 are all highly expressed in
lung airway epithelium in addition to other tissues such as the developing
endocardial cushions in the heart (Lu et
al., 2002; Wang et al.,
2004). However, there are important differences in the expression
patterns of Foxp1/2/4 in the lung and these differences may indicate specific
roles for each of these family members. Foxp1 is expressed in a polarized
fashion, with the highest level of expression in developing distal airway
epithelium and lower levels in more proximal airways. By contrast, Foxp2
expression is restricted to the distal airways, with little expression
detected in proximal airway epithelium by immunohistochemistry or in situ
hybridization. Foxp4 is expressed evenly throughout the airway epithelium with
no noticeable polarization along the proximal-distal axis. Foxp1 and Foxp4
cannot compensate for the loss of Foxp2 in the lung, as demonstrated by the
distinct alveolarization defects in Foxp2 mutant lungs and by the
lack of compensatory upregulation of Foxp1 or Foxp4. The extensive overlap in
expression of Foxp1/2/4 in tissues such as the lung, as well as their ability
to heterodimerize, suggest that a significant degree of redundancy might exist
and all three factors may regulate the same set of target genes in a
dose-dependent manner. Moreover, because AEC-1 cells are thought to derive
from AEC-2 cells, expression of Foxp2 in either cell type could contribute to
the alveolarization phenotype in Foxp2-/- animals.
The increased severity in lung-related defects in Foxp2-/-;Foxp1+/- mutants supports the hypothesis that Foxp1 and Foxp2 act in a cooperative and dose-dependent manner to regulate tissue-specific gene expression and development where these factors are co-expressed. The complete loss of both Foxp1 and Foxp2 in the lung will have to await the generation of a conditional Foxp1 allele, as Foxp1-/-;Foxp2-/- animals die prior to E11.5 (data not shown). Similar studies determining the role of Foxp1 and Foxp4 will also require conditional alleles owing to the early embryonic demise of compound mutants. These studies are the focus of future work and should reveal important dose-dependent functions in the developing lung as well as in other tissues, such as the cardiovascular system, where Foxp1/2/4 are co-expressed.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/10/1991/DC1
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ACKNOWLEDGMENTS |
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