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| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online December 12, 2006
doi: 10.1242/10.1242/dev.02720
1 Department of Medicine and University of Pennsylvania, Philadelphia, PA 19104,
USA.
2 Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104,
USA.
3 Laboratory of Metabolism, National Cancer Institute, National Institutes of
Health, Bethesda, MA 20892, USA.
4 Department of Cell and Developmental Biology, University of Pennsylvania,
Philadelphia, PA 19104, USA.
Author for correspondence (e-mail:
emorrise{at}mail.med.upenn.edu)
Accepted 27 October 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: GATA, Nkx, Transcription factor, Synergy, Lung, Mouse
|
INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The six known GATA factors in vertebrates can be divided into two
subfamilies; Gata1-3 and Gata4-6. All six GATA family members share a highly
conserved double zinc-finger DNA-binding domain. Gata1-3 are primarily
expressed in hematopoietic tissues, and are required for erythrocyte
differentiation (Gata1 and Gata2), T cell development (Gata3) and
hematopoietic stem cell development (Gata2) (reviewed in
Weiss and Orkin, 1995
).
Gata4-6 are expressed in heart and endodermal-derived tissues, such as the
lung and intestine, and all three members have been shown to play crucial
roles in the development of these tissues (reviewed in
Molkentin, 2000). In
particular, Gata6 can activate several lung epithelial-restricted genes,
including surfactant protein A (SP-A, also known as Sftpa1 -
Mouse Genome Informatics), surfactant protein C (SP-C, also known as
Sftpc - Mouse Genome Informatics) and Wnt7b, and is required
for proper lung epithelial differentiation in vivo
(Bruno et al., 2000;
Liu et al., 2002a;
Weidenfeld et al., 2002).
Members of the Nkx family of homeodomain transcription factors are
expressed in pulmonary epithelium and cardiomyocytes. Titf1 (also known as
Nkx2.1, and hereafter referred to as Nkx2.1) is expressed throughout the
conducting airway epithelium and both loss- and gain-of-function experiments
have demonstrated an essential role for this factor in lung development
(Kimura et al., 1996). Loss of
Nkx2.1 results in an early and severe block in branching morphogenesis of the
lung, as well as a severe loss of epithelial cell differentiation
(Kimura et al., 1996;
Minoo et al., 1999;
Yuan et al., 2000). In the
heart, Nkx2.5 is expressed in the developing bilateral precardiac mesoderm
early in development, starting at E7.5
(Lyons et al., 1995). Loss of
Nkx2.5 in mice results in embryonic demise at E9.5 resulting from aberrant
cardiac development with defects in looping morphogenesis and ventricular
specification (Lyons et al.,
1995). Dominant mutations in NKX2.1 and NKX2.5
in humans lead to congenital pulmonary and cardiac defects, respectively,
demonstrating their importance in adult tissue homeostasis
(Krude et al., 2002;
Schott et al., 1998).
The lung arises from an out pouching of the ventral foregut at
approximately E9.5 of mouse development. The primitive airways grow quickly in
an arborized fashion through branching morphogenesis. The lung is patterned in
a distinct proximal-distal manner and expression patterns of lung-restricted
genes define epithelial cell types within the developing lung. For example,
SP-C is expressed first at E10.5 in the distal tips of the growing airway
epithelium and later its expression is restricted to alveolar type-2 cells
(AEC-2) in the alveolus. By contrast, Clara cell 10 kd protein (CC10, also
known as Scgb1a1 - Mouse Genome Informatics) is expressed exclusively by Clara
cells lining the bronchioles and upper airways, beginning at approximately
E16.5. Transcription factors such as Gata6 and Nkx2.1 are also expressed in a
proximal-distal manner, with Gata6 being expressed at high levels in the
distal airway epithelium and later in AEC-2 cells while Nkx2.1 is expressed
throughout the developing airway epithelium, with highest levels in the distal
airway epithelium (Morrisey et al.,
1996; Yuan et al.,
2000).
Recent evidence demonstrates that GATA- and Nkx-family members physically
interact to synergistically activate target genes in lung, heart and vascular
smooth muscle in vitro (Liu et al.,
2002a; Nishida et al.,
2002; Sepulveda et al.,
2002; Weidenfeld et al.,
2002). Whether these physical interactions are required for proper
development of any of these tissues in vivo is unknown. To determine whether
GATA-Nkx interactions are required for the regulation of tissue-specific gene
expression and development in vivo, we generated Gata6-Nkx2.1 double
heterozygous (G6-Nkx DH) mice to assess the result of
haploinsufficiency of both genes on lung epithelial differentiation and
development. Gata6 and Nkx2.1 are the only known GATA and Nkx family members
expressed in lung epithelia, and both are essential for the proper development
of this tissue (Kimura et al.,
1996; Liu et al.,
2002b; Yang et al.,
2002). G6-Nkx DH embryos and mice exhibit specific
defects in lung epithelial differentiation, which cannot be accounted for by a
classic genetic epistatic relationship between the two genes, indicating that
the protein-protein interaction between GATA- and Nkx-family members is
crucial for tissue-specific gene regulation and cell differentiation in
vivo.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Morrisey et al., 1998). These
lines were maintained on a C57BL/6:CD-1 mixed background, as described
previously (Kimura et al.,
1996; Morrisey et al.,
1998) and were intercrossed to generate G6-Nkx DH embryos
and adult mice. Embryonic age was established by considering noon of the day
that the vaginal plug was observed as E0.5. Embryos were harvested by Cesarean
section. Genotyping was performed by PCR from genomic DNA isolated from either
yolk sac or tails of the fetuses and adult mice. The PCR conditions and oligos
for genotyping the Nkx2.1+/- mice have been previously
described (Niimi et al.,
2001). Genotyping for the Gata6+/- allele was
performed using the following oligonucleotides: forward
5'-CATTCCTCCCACTCATGATCTATAG-3', reverse
5'-GGTCACATTACAATTAAGAGCAGC-3'.
Histology
Dissected lungs (E18.5 and adult) were fixed in 4% paraformaldehyde for 48
hours. Tissues were then dehydrated in ethanol and embedded in paraffin.
Paraffin sections were cut at 5 µm and used for histochemical staining, in
situ hybridization and immunohistochemistry. Immunohistochemistry using CC10
(Santa Cruz T-18, 1:500), SP-C (Chemicon AB3786, 1:1000) and Bmp4 (Santa Cruz
N-16, 1:100) was performed as previously described
(Shu et al., 2005). In situ
probes for Gata6, Nkx2.1 and aquaporin-5 (Aqp5) have been previously described
(Shu et al., 2005;
Yang et al., 2002).
Oligonucleotides to generate the in situ probes for RBP-L (also known as
Rbpsuhl - Mouse Genome Informatics), stearoly-CoA desaturase 1 (Scd1),
dipeptidyl peptidase 4 (Dpp4) and napsin (also known as Napsa - Mouse Genome
Informatics) are: Scd1 forward 5'-GGTGCCAAACACTCAGTTCACTTG-3',
reverse 5'-TGTAATACGACTCACTATAGGGAGCCTCTTGACTATTCCCACTCG-3'; Dpp4
forward 5'-TCCAGAAGACAACCTTGACCATTAC-3', reverse
5'-TGTAATACGACTCACTATAGGGGGGGACAGGCATCCTTAGTTAGG-3'; RBP-L forward
5'-AAAGAGCAGGAAGGAGAGAGATAGC-3', reverse
5'-TGTAATACGACTCACTATAGGGTCACCACAGGCAGATAGACGC-3'; napsin forward
5'-ATCGCTTTAATCCCAAGGCCTTCC-3', reverse
5'-TGTAATACGACTCACTATAGGGGCCAACATCGCTCTGAAGAATC-3'. All reverse
oligonucleotides contain a T7 RNA polymerase recognition sequence used to
generate labeled cRNA probes. Periodic acid-Schiff (PAS) staining was
performed as previously described (Yang et
al., 2002). Additional details on histological methods can be
found at the University of Pennsylvania Molecular Cardiology Research Center
web site
http://www.uphs.upenn.edu/mcrc/.
All data are representative of at least five embryos of each genotype.
Lung morphometry
Mesenchymal thickness was calculated by capturing digital images at
200x magnification; overlaying grid lines in a vertical, horizontal and
diagonal fashion; and measuring the mesenchymal thickness on at least five
inter-alveolar regions per field of view. This was performed on ten fields of
view per sample and on four samples of each indicated genotype. The Student's
t-test was used to calculate the significance of the differences
between each group.
Electron microscopy
Lung tissue from the indicated mouse embryos (three samples for each
genotype) were fixed in 2% gluteraldehyde with 0.1 M sodium cacodylate (pH
7.4) for 72 hours at 4°C. Samples were further incubated with 2% osmium
tetroxide and 0.1 M sodium cacodylate (pH 7.4) for 1 hour at 4°C.
Ultrathin sections were stained with lead citrate and uranyl acetate and
viewed on a JEM 1010 microscope.
Microarray and quantitative PCR studies
RNA was isolated from E18.5 lungs from wild-type and G6-Nkx DH
littermates (three samples from each genotype). Total RNA was transcribed to
generate biotinylated cRNA to use as a probe for Affymetrix mouse 230A
GeneChips. Three chips each were hybridized for both wild-type and
G6-Nkx DH samples. Data from these arrays was normalized using
Microarray Suite 5.0 (MAS5, Affymetrix) and Significance Analysis of
Microarrays (SAM). Changes in gene expression of 2.0-fold and greater were
considered significant. Treeview software was used to generate the heatmap
(http://rana.lbl.gov).
Total RNA was isolated with Trizol and quantitative-PCR was performed using
the oligonucleotides listed in Table
3 with an Applied Biosystems 7900HT system and Syber green
reaction mixture, as previously described
(Lepore et al., 2005).
|
). The criterion for a match was P-value
cutoff of 2x10-4, corresponding to a probability of less than
one random match in every 5 kb of genomic sequence. These matches were further
filtered using human-mouse genomesequence alignments to focus our analyses on
promoter regions that showed evolutionary conservation. These criteria for
matching have been evaluated previously and were shown to accurately detect
experimentally verified binding sites with a low false-positive rate of one
random match in every 50 kb of genomic sequence searched
(Levy and Hannenhalli, 2002).
Because of similar DNA-binding sites, this methodology cannot distinguish
between the enrichment of different members of transcription factor families.
For example, `Gata-2' denotes enrichment of conserved GATA DNA-binding
sites.
Cell transfection studies
The mouse versus human comparisons of the Dpp4 and Scd1 genomic regions
were performed using the mVista genomic analysis software
(http://genome.lbl.gov/vista/index.shtml).
The indicated regions of the mouse Scd1 and Dpp4 genomic
regions were cloned into the pGL3promoter vector using the following
oligonucleotides: Dpp4 1250 bp region A enhancer forward
5'-TGGTACCGTGGTAACAGGTTACGGCAAAGTTAGC-3', reverse
5'-ATCTCGAGCCTTTCCCTCTAAACAATTGCAGTAAC-3'; Scd1 732 bp region A
enhancer forward 5'-TGGTACCAACAGTGTGGTCCCCAAGAAGCAG-3', reverse
5'-ATCTCGAGCACCACCCAGCCTGGCTTGGCAAC-3'; Scd1 466 bp region B
enhancer forward 5'-ATGGTACCTGACGCTGGACACCCAGACAT-3', reverse
5'-ATCTCGAGTGTTGGTTCCCAGGACAATCC-3'. The Gata6 and Nkx2.1
expression plasmids that were used have been previously described
(Weidenfeld et al., 2002).
NIH-3T3 cells were transfected with the indicated plasmids using Fugene 6 as
previously described (Weidenfeld et al.,
2002). All transfections were assayed after 48 hours. Luciferase
activity was determined using a commercially available kit (Promega). Reported
values are normalized to cells lacking Gata6- or Nkx2.1-expressionplasmids and
represent the average of three assays performed in triplicate ±
standard error of the mean (s.e.m.).
Phospholipid analysis
Lungs from wild-type and G6-Nkx DH-mutant mice (age 3-5 months)
were lavaged, and phospholipids levels were measured as previously described
(Atochina et al., 2000). Three
mice from each genotype were used in these studies. Briefly, bronchoalveolar
lavage (BAL) fluid was subfractionated into two surfactant fractions: the
biophysically active large-aggregate (LA) form and the biophysically inactive
small-aggregate (SA) form. Lavage fluid was centrifuged at 1000
g for 10 minutes at 4°C to remove cells. The cell-free
supernatant was recentrifuged at 20,000 g for 40 minutes at
4°C for separation of LA surfactant in the pellet and SA surfactant in the
supernatant fraction. The resulting LA pellets were resuspended in saline for
biophysical and biochemical characterization. LA- and SA-surfactant fractions
were analyzed for total phospholipid content by extraction of total
phospholipid and determination of inorganic phosphorus content with a modified
method of Bartlett (Itoh et al.,
1986).
Chromatin immunoprecipitation assays
Chromatin was made from E18.5 mouse lung tissue using a commercially
available kit (Upstate Biotechnology). Lung tissue was minced, fixed with 1%
formaldehyde and chromatin was sheared by sonication to an average length of
500-600 bp. The antibodies used for immunoprecipitation were as follows: Gata6
(Santa Cruz Biotech, C-20) and Nkx2.1 (Santa Cruz Biotech, H-190). Reverse
cross-linked immunoprecipitated chromatin was subjected to both standard PCR
and quantitative PCR on an ABI 7900 using Syber green and the oligonucleotides
listed in Table 2.
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|
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Morrisey et al., 1996). Both
Gata6 and Nkx2.1 expression is observed in distal airway epithelium, as well
as in more-proximal bronchiolar epithelium that expresses the
Clara-cell-marker gene product CC10 (Fig.
1C-H). This suggests that postnatal survival requires the combined
expression of Gata6 and Nkx2.1, probably through the regulation of lung airway
epithelial development.
|
;
Lee et al., 1997;
Yang et al., 2002). CC10
expression was dramatically reduced in G6-Nkx DH lungs
(Fig. 2L). These changes in
SP-C and CC10 expression were not observed in either
Gata6+/- or Nkx2.1+/- lungs
(Fig. 2B,C,E,F,H,I).
Quantitative RT-PCR (Q-PCR) confirmed the decrease in CC10 expression observed
by immunohistochemistry, whereas expression of SP-B, another crucial
surfactant component that is expressed after E15.5 in alveolar type 2 cells,
was not significantly altered (Fig.
3A). Expression of other putative target genes, such as SP-C and
Wnt7b, was decreased by approximately 50%
(Fig. 3A). In contrast to these
findings, SP-C, SP-B and CC10 expression are all severely attenuated or lost
upon loss of Gata6- or Nkx2.1-function in vivo
(Liu et al., 2002b;
Minoo et al., 1999;
Yang et al., 2002). Thus,
although G6-Nkx DH lungs exhibited defects associated with epithelial
immaturity, expression of aquaporin-5 and SP-B argues against a general
inhibition of lung maturation in G6-Nkx DH embryos due to the late
gestational onset of their expression.
The defects observed in G6-Nkx DH mice could be caused by a
genetic epistatic relationship between these two factors, or it could be due
to their reported physical interaction
(Liu et al., 2002a;
Weidenfeld et al., 2002). To
determine whether Gata6 and Nkx2.1 regulate the expression of each other,
Q-PCR was performed to determine the expression of Gata6 and Nkx2.1 in
wild-type, Gata6+/-, Nkx2.1+/- and
G6-Nkx DH lungs. Loss of either Gata6 or Nkx2.1 expression did not
alter the expression of the other gene
(Fig. 3B). This is supported by
our previous finding that loss of Gata6 activity does not lead to changes in
Nkx2.1 expression (Yang et al.,
2002). Thus, the cooperative relationship between Gata6 and Nkx2.1
is unlikely to be caused by classic genetic epistasis, but rather by the
physical interaction of the two factors.
|
|
Given the increased glycogen content in G6-Nkx DH lungs, phospholipid levels were determined on surviving G6-Nkx DH as well as wild-type, Gata6+/- and Nkx2.1+/- mice to determine whether these mice had altered surfactant levels. A significant and reproducible decrease of approximately 60% in phospholipid content in bronchioalveolar lavage fluid was observed in G6-Nkx DH adult mice (Fig. 5A). A small but significant decrease in phospholipid content was also observed in the Nkx2.1+/- mice (Fig. 5A). By contrast, lung-to-body weight ratios and tidal volumes were not appreciably changed in any of the mice (Fig. 5B, and data not shown). The decreased level of phospholipids correlates with the increased glycogen content observed at E18.5 in G6-Nkx DH embryos and supports the hypothesis that Gata6 and Nkx2.1 act synergistically to regulate surfactant production and processing. Assuming that the most severely affected animals died in the neonatal period, these data demonstrate that epithelial differentiation and surfactant production is exquisitely sensitive to regulation by Gata6 and Nkx2.1.
|
|
),
we found that GATA DNA-binding sites were enriched in the upstream regulatory
regions of the downregulated genes (see Fig. S1C in the supplementary
material). The overall lack of enrichment of Nkx-binding sites may reflect the
restricted region (1 kb) that was analyzed. However, Nkx-binding sites are
enriched in the subset of genes that are enriched in GATA DNA-binding sites
(see Fig. S1C in the supplementary material).
|
|
;
Wesley et al., 2005). RBP-L is
a transcription factor related to RBP-J (also known as Rbpsuh - Mouse Genome
Informatics), the downstream nuclear effector of the Notch signal-transduction
pathway (Minoguchi et al.,
1997). RBP-L expression is restricted to the lung and brain during
development (Minoguchi et al.,
1999). Stearoly-CoA desaturase 1 (Scd1) is an enzyme that converts
saturated fatty acids into monounsaturated fatty acids and is crucial for the
conversion of glycogen into phospholipids in the pulmonary surfactant
(Zhang et al., 2004). Napsin
(also known as Napsa - Mouse Genome Informatics) is an aspartyl protease known
to process SP-B and SP-C into their mature forms
(Brasch et al., 2003;
Ueno et al., 2004).
Remarkably, the expression of Dpp4, Scd1, RBP-L and napsin were all
dramatically downregulated in G6-Nkx DH mutants, as measured by Q-PCR
and in situ hybridization (Fig.
6A-C), indicating that synergism between Gata6 and Nkx2.1 is
required for their expression. Comparison of wild-type,
Gata6+/-, Nkx2.1+/- and G6-Nkx DH
samples showed that loss of Nkx2.1, but not of Gata6, leads to a small but
detectable decrease in expression of these target genes, whereas
G6-Nkx DH lungs have much more dramatic decreases in expression of
these four genes (Fig. 6A).
This suggests that Gata6 acts as a modifier of Nkx2.1 function, and that
Nkx2.1 DNA-binding sites may be required for synergistic interaction between
the two factors. Thus, the microarray data from the present studies allowed us
to identify a global network of genes in the lung whose expression is finely
controlled in a synergistic manner in vivo by Gata6 and Nkx2.1. The large
percentage of genes involved in surfactant production and processing
identified in these analyses suggests that the synergism between Gata6 and
Nkx2.1 is crucial to this overall gene-expression program in the lung.
Bmp4 expression is compromised in G6-Nkx DH lungs
Because the microarray experiment was performed on lungs from E18.5
embryos, the genes identified represent the later stages of lung development.
Given that Nkx2.1 and Gata6 are both expressed earlier in lung development, we
sought to determine whether expression of putative target genes known to be
regulated by both these factors were altered. Bmp4 is a crucial morphogen
expressed in the distal tips of the growing airways during early lung
development. Bmp4 and its receptor Bmpr1a are both important in the early
stages of lung epithelial differentiation and branching morphogenesis
(Eblaghie et al., 2006).
Moreover, Bmp4 has been shown to be a direct target of Gata6 and Nkx2.1
(Nemer and Nemer, 2003;
Zhu et al., 2004). To
determine whether Bmp4 expression was specifically altered in G6-Nkx
DH mutants, immunohistochemistry was performed on E12.5 embryos to assess Bmp4
expression. No difference was observed between wild-type,
Gata6+/- and Nkx2.1+/- embryos
(Fig. 7A-C). However, a
significant decrease in Bmp4 expression was observed in the airways
G6-Nkx DH mutants (Fig.
7D). Q-PCR using cDNA derived from E12.5 lungs confirmed a
decrease of almost 80% (Fig.
7E). These data suggest that the synergistic role of Gata6 and
Nkx2.1 is required for the expression of crucial target genes, such as Bmp4,
supporting a role for this synergy in the early stages of lung
development.
|
;
Ntambi et al., 1988).
Comparison of mouse and human genomic regions for these two genes shows that
there are several conserved regions both proximal and distal to the start of
transcription (Fig. 8A). Within
some of these regions, multiple conserved GATA- and Nkx2.1-DNA-binding sites
are found clustered together. By contrast, other regions contained only GATA-
or Nkx2.1-DNA-binding sites. To determine whether Gata6 and Nkx2.1 bound to
these conserved sites in vivo, chromatin immunoprecipitation (ChIP) assays
were performed using chromatin from E18.5 mouse lungs. ChIP showed that Gata6
and Nkx2.1 associate with specific regions within both of these genes
(Fig. 8B,C). In particular,
regions A and B in the Scd1 gene and region A in the Dpp4
gene showed high-level association with both Gata6 and Nkx2.1. Gata6 and
Nkx2.1 were not found associated with region C in Scd1 or regions B,
D, and E in Dpp4, despite all these regions containing highly
conserved Gata6- and/or Nkx2.1-DNA-binding sites. Nkx2.1, but not Gata6, was
found to associate with region C in Dpp4, even though this region
contains conserved Gata6 DNA-binding sites. Remarkably, Gata6 was found to
associate in region B of the Scd1 regulatory locus even though GATA
DNA-binding sites were not found in this region, indicating that Gata6 can
interact through other DNA-binding factors to associate with specific regions
of chromatin. Given the presence of Nkx2.1-binding sites and the ability of
Gata6 to bind to Nkx2.1 (Liu et al.,
2002a; Weidenfeld et al.,
2002), this interaction probably explains this association, as
well as the sensitivity of these genes to combined Gata6 and Nkx2.1
haploinsufficiency.
|
).
These data also indicate that this synergy occurs preferentially in enhancer
elements containing a single type of DNA-binding site, in this case Nkx2.1
sites, and that Nkx2.1 acts as a DNA-binding `platform' for further
interaction with Gata6, which acts as a modifier of Nkx2.1 function. Together,
these data demonstrate that Gata6 and Nkx2.1 synergistically regulate a
distinct subset of genes in vivo, including Scd1, Dpp4 and
Bmp4, that are crucial for lung epithelial differentiation,
surfactant expression and airway homeostasis. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Weidenfeld et al., 2002).
Despite these important observations, no data on the in vivo relevance of
these interactions has been reported. Although Gata6 and Nkx2.1 could directly
regulate the expression of one another, the data presented here and in
previous reports on the lack of an effect on the expression of Nkx2.1 in
dominant-negative Gata6-expressing lungs, as well as the high level of
expression of Gata6 throughout the developing foregut prior to Nkx2.1
expression, argues against this mechanism
(Liu et al., 2002b;
Yang et al., 2002). Thus, the
in vivo synergism between Gata6 and Nkx2.1 is probably caused by the physical
interaction between these two transcription factors, which leads to the
regulation of a subset of lung epithelial genes required for lung epithelial
development and homeostasis. Given the changes in expression of target genes
to Nkx2.1, but not to Gata6, haploinsufficiency, and that Gata6 and Nkx2.1 are
both associated with regions of chromatin that contain only Nkx2.1 DNA-binding
sites, our data suggest that Gata6 acts as modifier of Nkx2.1 function.
|
;
Lyons et al., 1995;
Sepulveda et al., 1998;
Sepulveda et al., 2002). Thus,
our findings may have important implications for the synergy between these
transcription factor families in other developmental systems. However, tissues
such as myocardium express multiple GATA- and Nkx-factors that probably act
redundantly, making it difficult to analyze a synergistic effect using genetic
experiments (Evans, 1999;
Molkentin, 2000). Given that
lung epithelium expresses a single Nkx- and GATA-factors, lung morphogenesis
is an ideal developmental process in which to study such synergistic
interactions. In addition to redundancy, there may be specific differences in
how GATA- and Nkx-factors work in other tissues. GATA factors have been shown
to act directly upstream of Nkx2.5 in myocardium and Nkx2.5 has been shown to
be crucial for Gata6 expression in the heart, identifying a self-enforced
regulatory loop for these factors (Lien et
al., 1999; Molkentin et al.,
2000). Based on the data in this report, as well as on that from
previous studies, Gata6 and Nkx2.1 do not appear to act in such a manner in
the lung (Liu et al., 2002b;
Yang et al., 2002).
Recent data has demonstrated that a host of factors are required for proper
lung epithelial differentiation, including Foxa1, Foxa2 and C/EPB
(Cebpa) (Martis et al., 2006;
Wan et al., 2005;
Wan et al., 2004). We show
that haploinsufficiency in both Gata6 and Nkx2.1 leads to a disruption in a
genetic program required for Bmp4 expression in early lung development, as
well as a disruption in surfactant production later in lung development. Bmp4
has been shown to be a direct target of Gata6 and Nkx2.1, and its
downregulation in G6-Nkx DH lungs suggests that the synergistic
interaction between Gata6 and Nkx2.1 is required for its full expression
(Nemer and Nemer, 2003;
Zhu et al., 2004). These data
indicate that Gata6-Nkx2.1 interactions are crucial from the earliest stages
of lung development. Interestingly, the combined activity of Gata6 and Nkx2.1
is required for the expression of a set of genes important for surfactant
production and homeostasis, including Scd1 and Dpp4. Both
Gata6 and Nkx2.1 directly associate with conserved genomic regulatory regions
in Scd1 and Dpp4 in vivo, and directly activate, in some
cases synergistically, conserved upstream regulatory regions in each gene.
This synergy appears to occur only in enhancer elements containing Nkx2.1
DNA-binding sites, suggesting the differential regulation and usage of such
crucial protein-protein interactions. Such interactions could increase the
affinity of Gata6 and Nkx2.1 for additional co-factors or could increase the
affinity of the binding of Nkx2.1 to DNA, as has been suggested in
Gata4-Nkx2.5 interactions (Sepulveda et
al., 1998). The identification of Scd1 and Dpp4
as direct targets of Gata6- and Nkx2.1-regulation provides novel information
on how these transcription factors regulate both phospholipid production and
airway-epithelial homeostasis.
Defects in saccular development and surfactant production are common in
human neonates with bronchopulmonary dysplasia. Our finding that Gata6 can act
as a modifier of Nkx2.1 may help to explain why some human patients with
NKX2.1 mutations have respiratory defects whereas others appear
normal (Krude et al., 2002;
Pohlenz et al., 2002). The
gene-expression defects uncovered in G6-Nkx DH embryos and mice
represent targets that are specifically regulated by the synergistic activity
of these two transcription factors. Further studies into this and other
combinatorial interactions between GATA- and Nkx-factors in vivo will
undoubtedly reveal novel insights into the gene-gene and protein-protein
interactions described in other tissues.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02720/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Atochina, E. N., Beers, M. F., Scanlon, S. T., Preston, A. M.
and Beck, J. M. (2000). P. carinii induces selective
alterations in component expression and biophysical activity of lung
surfactant. Am. J. Physiol. Lung Cell. Mol. Physiol.
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