Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart
Bin Zhou1,*,
Bingruo Wu1,
Kevin L. Tompkins1,
Kathleen L. Boyer1,
Justin C. Grindley1 and
H. Scott Baldwin1,2
1 Department of Pediatrics, Vanderbilt University School of Medicine, Nashville,
TN 37232, USA
2 Department of Cell and Developmental Biology, Vanderbilt University School of
Medicine, Nashville, TN 37232, USA
*
Author for correspondence (e-mail:
bin.zhou{at}vanderbilt.edu)
Accepted 13 December 2004
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SUMMARY
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Nfatc1 is an endocardial transcription factor required for development of
cardiac valves. Herein, we describe identification and characterization of a
tissue-specific enhancer in the first intron of murine Nfatc1 that
activates a heterogenic promoter and directs gene expression in a
subpopulation of endocardial cells of the developing heart: the pro-valve
endocardial cells. This enhancer activity begins on embryonic day (E) 8.5 in
endocardial cells at the ventricular end of the atrioventricular canal,
intensifies and extends from E9.5 to E11.5 in endocardium along the
atrioventricular canal and outflow tract. By E12.5, the enhancer activity is
accentuated in endocardial cells of forming valves. Sequential deletion
analysis identified that a 250 bp DNA fragment at the 3' end of the
intron 1 is required for endocardial-specific activity. This region contains
two short conserved sequences hosting a cluster of binding sites for
transcription factors, including Nfat and Hox proteins. Electrophoresis
mobility shift and chromatin immunoprecipitation assays demonstrated binding
of Nfatc1 to the Nfat sites, and inactivation of Nfatc1 downregulated the
enhancer activity in pro-valve endocardial cells. By contrast, mutation of the
Hox site abolished its specificity, allowing gene expression in non pro-valve
endocardium and extracardiac vasculature. Thus, autoregulation of Nfatc1 is
required for maintaining high Nfatc1 expression in pro-valve endocardial
cells, while suppression through the Hox site prevents its expression outside
pro-valve endocardial cells during valve development. Our data demonstrate the
first autonomous cell-specific enhancer for pro-valve endocardial cells and
delineate a unique transcriptional mechanism that regulates endocardial Nfatc1
expression within developing cardiac valves.
Key words: Mouse, Heart, Endocardium, Nfatc1, Transcription, Enhancer
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Introduction
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Previous studies suggest that pro-valve endocardial cells (endocardial
cells lining of developing cardiac valve) are a unique subpopulation of
endocardial cells with a distinct developmental potential
(Wunsch et al., 1994
). During
embryogenesis and in response to local myocardial cues, endocardial cells in
the cardiac outflow tract (OFT) and atrioventricular canal (AVC) undergo an
endocardial-to-mesenchymal transformation (EMT), a morphogenic process
required for cardiac valve formation and chamber septation
(Barnett and Desgrosellier,
2003
; Eisenberg and Markwald,
1995
; Schroeder et al.,
2003
). Despite this, little is known about the molecular
mechanisms that establish this unique pro-valve endocardial subpopulation
during cardiac ontogeny in vivo.
Members of the nuclear factors of activated T cell (Nfat) family, which
mediate transcriptional responses of the Ca2+/calmodulin-dependent
protein phosphatase calcineurin, have been implicated in cardiovascular
development (Bushdid et al.,
2003
; Graef et al.,
2001
) and cardiac hypertrophy
(Antos et al., 2002
;
Molkentin et al., 1998
;
Wilkins et al., 2002
). Nfatc3
and Nfatc4 are involved in the development of normal myocardium
(Bushdid et al., 2003
), and
patterning the vasculature in early mouse embryos
(Graef et al., 2001
). Nfatc3
is also required for cardiac hypertrophic response in vivo
(Wilkins et al., 2002
). We and
others have studied the function of Nfatc1, in the mouse by genetic
inactivation, and have found that it is required for cardiac valve formation
(de la Pompa et al., 1998
;
Ranger et al., 1998
).
Consistent with a function in formation of these endocardial-derived
structures, Nfatc1 is exclusively expressed in the endocardium from the
initiation of endocardial differentiation in the primary heart-forming field.
Subsequently, there is accentuation and sustained expression in pro-valve
endocardial cells during EMT and early valve formation followed by rapid
attenuation at the initiation of valve leaflet remodeling
(Chang et al., 2004
;
de la Pompa et al., 1998
).
Nfatc1 is thus a cell-type-specific transcription factor prevalent in
pro-valve endocardial cells and represents a unique candidate for delineating
the molecular control of endocardial gene transcription during EMT and cardiac
valve development.
In this study, we report the identification and characterization of an
autonomous cell-specific transcriptional enhancer for pro-valve endocardial
cells. Located in the first intron of the mouse Nfatc1, this 250 bp
enhancer sequence contains a cluster of Nfat sites and a single Hox site,
which are required for gene expression exclusively in pro-valve endocardial
cells of the OFT and AVC during valvulogenesis. We demonstrate that
autoregulation of Nfatc1 is essential for maintaining the enhancer activity in
pro-valve endocardial cells, while the Hox site is required for suppressing
its activity in tissues outside of pro-valve endocardium. Our study suggests
that this dual regulation provides a molecular mechanism for restricted and
high level expression of Nfatc1 in pro-valve endocardial cells, where Nfatc1
is essential for cardiac valve development.
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Materials and methods
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Transgenic reporter constructs
The mouse bacterial artificial chromosome (BAC) clone containing the murine
Nfatc1 was isolated from a 129/SvJ BAC library
(Zhou et al., 2002
). In vivo
lacZ promoter reporter constructs were based on pWhere (Invivogen).
The promoter reporter construct NX-lacZ consists of a 6.3 kb
NheI-XhoI P1 promoter of the murine Nfatc1
(Zhou et al., 2002
), and
XS-lacZ contains a 4.5-kb XhoI-SacII intronic P2 promoter,
starting 76 nucleotides downstream of the 3' end of exon 1 and ending 77
nucleotides into exon 2 (Fig.
2A). The enhancer reporter constructs used a heat-shock protein
minimal promoter, HSP68, which, by itself, does not produce detectable
activity in vivo. The parent enhancer reporter construct,
BB-HSP-lacZ, contained a 4.1 kb BssHII-BssHII
intron 1 fragment, starting 214 nucleotides downstream of the 3' end of
exon 1 and ending 199 nucleotides upstream of the 5' end of exon 2
(Fig. 2A). Serial 5'
deletions of BB-HSP-lacZ were achieved by insertion of PCR-amplified
DNA fragments into the pWhere-HSP vector, yielding an additional seven
constructs, named d1 to d7 (Fig.
4A-C). For mutation constructs, a PCR-based mutagenesis was
performed as described before (Zhou et al., 1998). Individual mutation of core
nucleotides for Gata, or Hox- or Smad-binding sites, was introduced into the
conserved putative cis-enhancing elements in d5 construct. All mutations were
confirmed by sequence analysis.

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Fig. 2. (A) Schematic diagram of the Nfatc1 promoter/enhancer reporter
constructs. (B) Transient transgenic embryos following X-gal staining
documents that the 6.2-kb NheI-XhoI P1 promoter-reporter
(NX-lacZ) does not produce endocardial gene expression at E11.5. However, the
BB-HSP-lacZ enhancer-reporter, containing 4.1 kb
BssHII-BssHII fragment of the P2 (intron 1) regulatory
region, linked to the HSP68 minimal promoter, is able to drive expression
specifically in the endocardial lumen of the atrioventricular canal (AVC) and
in the conal (c) and truncal (t) regions of the developing outflow tract. No
expression is detected in the atrium (a) or ventricle (v) or in the
extracardiac vasculature. (C) Table summaries this group of transgenic
experiments. TG, transgenic embryos; ECS, endocardial-specific expression; ET,
ectopic expression.
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Fig. 4. Analysis of ß-galactosidase activity in embryos from four independent
stable transgenic lines shows consistent pro-valve endocardial enhancer
activity of the 4.1 kb BssHII-BssHII P2 fragment in
whole-mount-stained E8.5 to E12.5 embryos (A-E). X-gal staining is restricted
to the endocardial cells in AVC (arrow) and OFT (arrowhead), especially
intensified at later stages in the regions of forming valves and septa. (F-J)
Sectioning of stained E8.5, E9.5, E12.5 and E14.5 embryos highlights this
enhancer activity for the pro-valve endocardial cells of the forming valves.
ao, aorta; av, aortic valve; pt, pulmonary trunk; pv, pulmonary valve; la,
left atrium; ra, right atrium; lv, left ventricle; rv, right ventricle; mv,
mitral valve; tv, tricuspid valve.
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Transgenic mice
Transgenic fragments were separated from the plasmid vector by
electrophoresis of PacI-digested plasmid and purified using Qiaex II
(Quiagen). The purified DNA fragments were dissolved in the injection buffer
(10 mM Tris HCl, pH 7.5 and 0.1 mM EDTA) at a concentration of 2.5 µg/ml.
DNA was then injected into the pronucleus of 0.5-day-old fertilized C57BL/6
eggs and the eggs were transferred into the oviducts of ICR pseudo-pregnant
foster females. Embryos were harvested, stained with X-gal to detect
ß-galactosidase activity and yolk sacs were processed for PCR genotyping.
Transgenic lines were established by breeding B6D2F1 mice with PCR-positive
founders, and embryos were harvested at embryonic day (E) E8.5 to 14.5.
ß-Galactosidase detection in whole embryos
Embryos were collected in PBS, fixed in 4% paraformaldehyde, and stained in
X-gal solution overnight at 30°C. The stained embryos were cleared in a
gradient of glycerol and photographed in 100% glycerol with a dissecting
photomicroscope. Whole-mount-stained embryos were then processed for sectional
examination. For sections, stained embryos were post-fixed with 4%
paraformaldehyde, dehydrated in ethanol, cleared in xylene and embedded in
paraffin. Continuous cross-sections of 6 µm thickness were cut,
counterstained with Eosin and mounted in Permount.
Primary endocardial cell cultures
We isolated and established primary embryonic endocardial cell cultures
from E11.5 hearts using a magnet-based antibody affinity protocol
(Marelli-Berg et al., 2000
).
Briefly, hearts (without large arteries and surrounding tissues) of E11.5
embryos from 10 pregnant ICR animals were dissected and digested with
collagenase (Sigma). Single cell suspensions in PBS plus 2% FBS were incubated
with anti-Pecam1 and anti-endoglin monoclonal antibodies, and
biotinylated-isolectin B-4. Endocardial cells were then isolated using
magnetic bead-conjugated secondary antibodies and magnetic bead-conjugated
avidin, seeded into one-well of a 24-well plate with irradiated OP9 feeder
cells, and cultured with M199 plus 20% FBS for a week. Confluent cells were
then split into one gelatinized well of a 12-well plate without feeders. Using
this method, we obtained enriched (>80% pure) endocardial cell cultures
determined by their nuclear presence of Nfatc1. These cells express various
endothelial markers including Pecam1/CD31, Tie2, endoglin/CD105 and
VE-cadherin, and maintain their endocardial phenotype and morphology over
10-15 passages (one to four splits per passage).
Electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays
Preparation of nuclear extracts from primary cultured endocardial cells and
subsequent EMSA were performed as described before
(Zhou et al., 2002
). ChIP
assays were carried out using commercially available reagents and protocol
from Upstate (Lake Placid, NY) and monoclonal anti-Nfatc1 specific antibodies
(7A6) according to manufacturer's protocol. The primer sets for PCR
amplification include: 5' ChIP primer (5' GGAGAAAAGCAGCCATTGAAAC
3') and 3' ChIP primer (5' CTGAGTAGGTGCTGGGTGTGAC 3'),
which give a 404 bp DNA product containing two conserved regions with multiple
Nfat sites; and 5' control primer (5' GGCCAGGAGCGACGCGGACGAAG
3') and 3' control primer (5' GAGAAAATGAAAGACAGCAAGATAG
3'), which generate a 426 bp product without consensus Nfat sites.
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Results
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P1 promoter of Nfatc1 is activated in endocardial cells of the developing heart
Two Nfatc1 promoters, P1 and P2, have been described, which in the mouse T
cells direct the synthesis of Nfatc1.
and Nfatc1.ß isoforms,
respectively. To investigate the function of the P1 and P2 promoters in the
developing heart, we cloned two DNA fragments, a 6.3 kb
NheI-XhoI P1 promoter region and a 4.5 kb
XhoI-SacII P2 promoter region
(Fig. 1A). Using an RT-PCR
strategy with a set of three isoform-specific primers
(Fig. 1B), we observed the
presence of both isoforms in cultured primary E11.5 endocardial cells.
However, consistent with the previous report that the P1 promoter activity
accounts for over 90% Nfatc1 transcripts in T cells
(Chuvpilo et al., 2002
),
Nfatc1.
was abundant in the endocardial cells as its transcripts were
easily detected with a 35-cycle of amplification while the transcripts of
Nfatc1.ß regulated by the P2 promoter were barely detected using 40
cycles of amplification. Similar findings were obtained from mRNA isolated
from E11.5 embryonic hearts (data not shown).
The first intron of the murine Nfatc1 directs endocardial-specific gene expression during heart development
We next examined whether the 6.3-kb NheI-XhoI P1 promoter
contains the essential regulatory elements required for endocardial-specific
expression in vivo (Fig. 2A).
Although the 6.3-kb NheI-XhoI P1 promoter was able to drive
accentuated endothelial-specific gene expression in vitro (data not shown),
our transient transgenic analysis in mouse demonstrated that the
NX-lacZ reporter with the 6.3-kb NheI-XhoI P1
promoter was unable to confer detectable endocardial expression in vivo
(Fig. 2B). We also examined the
4.5 kb XhoI-SacII P2 promoter in transient transgenic
experiments (Fig. 2A).
Consistent with the RT-PCR results (Fig.
1C), which indicated that the P2 region is at best a weak
promoter, the XS-lacZ reporter with the 4.5 kb
XhoI-SacII P2 promoter failed to drive endocardial gene
expression (data not shown).
Finding that the Nfatc1.
is the major transcript detected in the
endocardium during embryogenesis but that the P1 promoter and its upstream
sequences were insufficient to drive detectable endocardial expression, we
reasoned that enhancers outside the NheI-XhoI fragment must
contribute to P1 activation. In scanning the mouse Nfatc1 locus for
the putative enhancers, we observed that four out of eight conserved domains
(mouse/human) in the 10.8 kb NheI-SacII fragment were
located within intron 1, proximal to the P2 minimal promoter. Therefore, a 4.1
kb BssHII-BssHII fragment, without the P2 minimal promoter,
was tested for the enhancer activity using the HSP68 promoter, and the
enhancer reporter was named BB-HSP-lacZ
(Fig. 2A). When this construct
was used to produce transgenic embryos, X-gal staining revealed strong
ß-galactosidase activity in the endocardial lumen of atrioventricular
canal (AVC) (arrowhead) and outflow tract (OFT) (arrow) of the heart
(Fig. 2B). Nine out of 10 X-gal
stained transient transgenic embryos exhibited endocardial-specific expression
with no staining in the myocardium or in the endothelium outside the heart
(Fig. 2C).
Further transient transgenic analysis indicated that this
endocardial-enhancer activity was detectable at E9.5 by whole-mount X-gal
staining (Fig. 3A),
highlighting the lumen of AVC (arrowhead), and marking the proximal OFT (the
broken line indicates the border of the distal and the proximal OFT).
Sectioning of the whole-mount-stained E10.5 embryos confirmed the endocardial
specificity of this enhancer (Fig.
3B). Importantly, the enhancer was only activated in the pro-valve
endocardial cells that overlie the forming endocardial cushions in the AVC
(arrowhead) and the proximal OFT (arrow). The enhancer activity was not found
in those transformed endocardial cells that were invading the extracellular
matrix-rich endocardial cushions. By E11.5
(Fig. 3C), the endocardial
activity of the enhancer was persistent in the AVC (arrowhead) and extended
from the proximal to distal part of the OFT (arrow), but was continuously
inactivated in the mesenchymal cushion cells derived from transformed
endocardial cells.

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Fig. 3. X-gal staining in transient transgenic embryos shows endocardial-specific
enhancer activity of the 4.1 kb BssHII-BssHII P2 fragment in
whole-mount E9.5 embryos (A) and sections of whole-mount-stained E10.5 (B) and
E11.5 (C) embryos. ß-Galactosidase activity is restricted to the
pro-valve endocardial cells in AVC (arrowhead) and OFT (arrow). The enhancer
is not activated in the mesenchymal cells derived from transformed endocardial
cells in the AVC and OFT endocardial cushions. Top row, OFT; bottom row, AVC.
a, atrium; c, conus; t, truncus; v, ventricle.
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To confirm the finding of transient transgenic analysis and to study in
detail the temporal and spatial activity of this endocardial-enhancer, four
independent transgenic mouse lines were established with the
BB-HSP-lacZ reporter construct.
Fig. 4 shows representative
findings of endocardial enhancer reporter activity during cardiogenesis.
Expression was first detectable at E8.5
(Fig. 4A), where lacZ
expression was observed in few isolated cells located at the ventricular
entrance of the AVC (arrowheads). By E9.5
(Fig. 4B), well-defined
endocardial-specific expression of lacZ was seen in the AVC of
whole-mount-stained embryos (arrowhead). At E10.5
(Fig. 4C), lacZ
expression extended into the proximal region of OFT (arrow). At E11.5
(Fig. 4D),
ß-galactosidase-positive endocardial cells were evident in the septating
OFT (arrow) and AVC (arrowhead), with expression accentuated at the proximal
ventricular region of the forming aortic and pulmonary trunk as well as at the
ventricular ends of AVC. By E12.5 (Fig.
4E), the endocardial staining was intensified in the newly
septated OFT and AVC, as well as the valvulogenic areas. Sectioning of
whole-mount-stained embryos indicated that the enhancer is initially
activated, between E8.5 or E9.5, in the endocardial cells of the AVC and OFT
(Fig. 4F,G); and that its
activity increases reaching maximal expression at E12.5 in the endocardial
cells lining AVC and recently separated OFT (aortic and pulmonary outlets)
(Fig. 4H). In particular,
activity was concentrated in those endocardial cells of the forming cardiac
valves, marking only the pro-valve endocardium at the endocardial-endothelial
junction of ventricular outlets and distal arterial root
(Fig. 4I). By E14.5 when the
remodeled endocardial cushions begin to assume the morphology of discrete
valve leaflets, the enhancer activity was significantly diminished and
detectable only in a few endocardial cells lining the newly formed valve
leaflets (Fig. 4J).
A 781 bp sequence in the P2 regulatory region is required for the endocardial-specific gene expression in the developing heart
Comparative sequence analysis of the first intron of the mouse and the
human revealed that, in addition to the conservation of the proximal (core) P2
promoter region, there were two distal highly conserved regions located in the
4.1-kb BssHII-BssHII intron 1 fragment
(Fig. 5A). Furthermore, a 211
bp pyrimidine-rich stretch and 10 copies of a CTTTT repeat were found in this
intron fragment. Using PCR cloning, nucleotide sequences in this P2 fragment
essential for endocardial-specific expression were determined by systematic
and sequential removal of these unique sequences to produce deletions of the
BB-HSP-lacZ reporter construct, named d1-d7
(Fig. 5A). Analysis of this
series of deletion constructs by transient mouse transgenesis and whole-mount
staining of E11.5 embryos demonstrated that constructs d1-d5 were able to
confer endocardial-specific expression identical to that of the parent
BB-HSP-lacZ reporter (Fig.
5B). Thus, the d5 deletion construct containing a 1.5 kb 3'
end sequence of intron 1 is sufficient for the endocardial-specific
expression. Removal of 781 bp sequence between the 200 bp conserved region and
CTTTT repeats completely abolished this endocardial-specific activity,
indicating the d6 sequence with the CTTTT-repeats alone is insufficient for
the endocardial gene expression. The endocardial specificity of the d5
construct was verified by cross-sectional examination of the
whole-mount-stained embryos (Fig.
5C). At the single cell level of resolution, the expression of
lacZ is exclusively restricted to the endocardial cells in the OFT
(arrow) and AVC (arrowhead). ß-Galactosidase activity is extinguished in
those cells that have undergone mesenchymal transformation and invaded the
matrix-rich cushions, and no ß-galactosidase activity was found in the
endothelial cells outside of the heart. These data clearly demonstrated that a
crucial enhancer sequence for the pro-valve endocardial cells is located
within the 781 bp region.

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Fig. 5. Deletional analysis of the endocardial enhancer. (A) Schematic depicting
the unique features of the 4.1 kb BssHII-BssHII intron 1
region and the deletional reporter constructs (d1-d7). (B) Representative
whole-mount staining of E11.5 embryos demonstrates the presence of the
pro-valve endocardial enhancer activity in d1-d5 but not d6 and d7. (C)
Cross-sectional analysis of d5 transgenic embryos (E11.5) shows that the
enhancer activity is exclusively restricted to the pro-valve endocardial cells
in AVC (arrow) and OFT (arrowhead). The enhancer is not activated in the
transformed cells of the endocardial cushions and endothelial cells outside of
the heart.
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The 781 bp sequence is sufficient for the endocardial-specific gene expression in the developing heart
Together, the data revealed that transcription of Nfatc1 in the pro-valve
endocardium is positively regulated by cis-acting elements located in the 781
bp intron 1 sequence (Fig. 6A).
To further determine whether this region was sufficient, in isolation, to
direct pro-valve endocardial-specific expression, the 781 bp sequence was
cloned into the pWhere-HSP reporter and the construct was named ECE
(endocardial cell enhancer)-HSP-lacZ
(Fig. 6B). An additional
construct (ECE
-HSP-lacZ) was generated by PCR cloning to
remove 250 bp of 5' end sequence that contained two short conserved
domains, ECE1 and ECE2, harboring a cluster of putative transcriptional
binding sites (Fig. 6A). The
pro-valve endocardial enhancer activity of these constructs was then evaluated
in transient transgenic analysis. Whole-mount-stained E11.5 embryos
(Fig. 6C,D) and hearts
(Fig. 6E,F) with the
ECE-HSP-lacZ construct demonstrated the expression of lacZ
reporter at the lumen of both OFT (Fig.
6, arrowhead) and AVC (Fig.
6, arrow) region. Thus, the 781 bp ECE was sufficient for
pro-valve endocardial-specific expression, and functioned as an autonomous
tissue-specific enhancer. Deletion of the 250 bp sequence containing ECE1 and
ECE2 completely abolished activity (data not shown), indicating that a crucial
cis-enhancer element(s) is contained in this 250 bp sequence.

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Fig. 6. (A) The activation of P1 promoter by the 781 bp sequence of the P2
regulatory region. Two short conserved elements (mouse/human) are located in
this sequence with a cluster of binding sites for known transcription factors.
(B) A schematic shows deletional analysis of the conserved putative
endocardial enhancers (ECEs). (C-F) Whole-mount staining of E11.5 embryos
(C,D) or isolated hearts (E,F) demonstrates that the ECE-HSP-lacZ,
but not the ECE -HSP-lacZ reporter (data not shown), is
sufficient to direct gene expression specifically in the pro-valve endocardial
cells of forming valves and septa. ao, aorta; pt, pulmonary trunk; la, left
atrium; ra, right atrium; lv, left ventricle; rv, right ventricle; avc,
atrioventricular canal; arrow, AVC; arrowhead, OFT.
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The 250 bp Nfat site-rich region directs autoregulation of Nfatc1 expression during valve formation
The presence of multiple Nfat sites in ECE1 and ECE2 of the 250 bp sequence
prompted us to examine whether Nfatc1 expression is required for activation of
the enhancer as previously described for its P1 promoter in T cells
(Chuvpilo et al., 2002
;
Zhou et al., 2002
). We
therefore crossed the BB-HSP-lacZ transgenic reporter line into the
previously described Nfatc1-null mutant mouse line
(Ranger et al., 1998
). We
found that, in both whole-mount E11.5 embryos and cross-sections
(Fig. 7), there was a
consistent reduction or absence of endocardial gene expression in both OFT
(arrow) and AVC (arrowhead) of Nfatc1-null embryos
(Fig. 7D-F) compared with their
heterozygous littermates (Fig.
7A-C).

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Fig. 7. Autoregulation of Nfatc1 enhancer activity. The
BB-HSP-lacZ reporter transgenic line was crossed into the existing
Nfatc1-null mutant line. Compared to the heterozygous littermates (A-C),
inactivation of Nfatc1 greatly reduced the pro-valve endocardial enhancer
activity of the BB-HSP-lacZ reporter construct (D-F). A consistent
reduction of endocardial lacZ expression is shown in the OFT (arrow)
and AVC (arrowhead) of the E11.5 heart when crossed into Nfatc1-null
background
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We further analyzed whether Nfatc1-dependent expression was dependent on a
direct interaction of Nfatc1 and the Nfat sites in ECE1 and ECE2 in situ. This
required that we develop a new primary embryonic endocardial cell culture from
E11.5 embryos as isolation of a sufficient number of endocardial cells
directly from wild-type and Nfatc1-null mutant embryos was not technically
feasible. As shown in Fig. 8,
these primary endocardial cells (ECCs) have a uniform endothelial-like
morphology and express nuclear localized Nfatc1
(Fig. 8A), as well as multiple
endothelial cell markers, such as Tie2, Pecam1/CD31, endoglin/CD105 and
VE-cadherin (data not shown). We then performed EMSA analysis using nuclear
extracts prepared from these primary ECCs. Three oligonucleotide probes (N1,
N2 and N3) were generated with or without the deletion of core dinucleotides,
GG or CC, that are characteristic of the Nfat-binding site; mutated probes
were named N1
, N2
1, N2
2 and N3
(Fig. 8B). EMSA showed that
protein-DNA binding complexes formed using N1 or N2
(Fig. 8C, arrow) but not N3
probe (data not shown). Importantly, mutation of any Nfat site in N1 or N2
greatly abolished formation of Nfatc1-DNA complexes, suggesting that these
Nfat sites are functional in situ. We then performed ChIP assays with
chromatin prepared from cultured wild-type or Nfatc1-null endocardial cells in
which the Rel-homology DNA-binding domain has been deleted
(Ranger et al., 1998
). A set
of primers (Fig. 8B, arrow)
encompassing ECE1 and ECE2 were used to amplify chromatin pulled down by the
anti-Nfatc1-specific monoclonal antibody 7A6. The results demonstrate that
Nfatc1 binds to this DNA fragment in situ
(Fig. 8D), whereas controls
using either no antibody or chromatin from Nfatc1-null endocardial cells
showed no product after PCR amplification. In addition, irrelevant primers
that flank a non-Nfat site fragment DNA did not produce a PCR product (data
not shown). Together, these data suggest that Nfatc1 expression is
autoregulated during valve formation by interaction of Nfatc1 and the 250 bp
Nfat site-rich region.

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Fig. 8. (A) Primary culture of embryonic (E11.5) endocardial cells (ECCs). ECCs
form a colonized monolayer surrounded by fibroblastic-like OP9 feeder cells at
passage 1 (p1). At p3, ECCs exhibit uniform, endothelial-like morphology with
a typical `cobblestone' appearance. Approximately 80% of the ECCs expresses
nuclear localized Nfatc1 (green; negative cells are indicated by arrowheads in
DAPI staining). Negative control (no primary antibody) is shown in ECC(-Ab).
(B) The 781 bp enhancer region. Two short stretch conserved sequences, ECE1
and ECE2, are shown with Nfat-binding sites highlighted in red and deleted GG
or CC bi-nucleotides of core binding site are underlined. The arrows indicate
the location of primers for the ChIP assays. (C) EMSA demonstrating that
mutation of the Nfat-binding sites in either the N1 or N2 regions results in
attenuation of Nfatc1 binding (arrows). (D) Results of the ChIP assay document
PCR amplification of the chromatin region encompassing ECE1 and ECE2 from
wild-type ECCs following immunoprecipitation with an Nfatc1 antibody (7A6)
(arrow) and absence of chromatin amplification without antibody or using
cultured Nfatc1-null ECCs.
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The Hox site is required for suppression of the enhancer activity in non pro-valve endocardial cells
To determine the function of other transcription factor-binding sites
adjacent to the Nfat sites, constructs containing deletion of core nucleotides
for Gata, Hox and Smad sites were used in transient transgenic assays.
Although mutation of the Gata and Smad sites did not alter the specificity of
in vivo lacZ expression (data not shown), mutation of the Hox site
resulted in activation of the enhancer in endocardial cells outside of the
valve forming region or non pro-valve endocardial cell and vascular bed
outside of the heart (Fig. 9).
In whole-mount-stained E11.5 and E12.5 embryos, activity of the enhancer was
readily observed in umbilical cord, intersomitic
(Fig. 9A, arrows) and head
vasculature (Fig. 9C). Sectional analysis confirmed that the enhancer is activated in the endothelium
of peripheral vasculature, such as in the head
(Fig. 9C, inset), and
endothelial cells of ductus venous of E12.5
(Fig. 9F, arrow) and E11.5
embryos (Fig. 9H, arrowhead in
inset). Within the heart, aberrant enhancer activity was observed in the
endocardial cells of the trabeculated ventricular outlet in E12.5
(Fig. 9E, arrowhead in the
inset) and E11.5 hearts (Fig.
9G, marked by a star), and sinus venous valves
(Fig. 9H, arrow). The
dysregulated enhancer activity was also observed in the cushion mesenchymal
cells (Fig. 9E, inset). In
addition to its abnormal non pro-valve endocardial activity, activity of the
mutated enhancer appeared to be increased in the pro-valve endocardial cells
(Fig. 9B,D, arrowheads),
although the transient transgenic approach did not allow us to compare
relative activity between the wild-type and mutated enhancer. Thus, these data
suggested that the Hox site is required for maintaining the pro-valve
endocardial specificity of the enhancer by suppression of its activity in non
pro-valve endocardial tissues.

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|
Fig. 9. Whole-mount (A-D) and section (E-H) analysis reveals that mutation of the
Hox site results in activation of the enhancer outside the pro-valve
endocardial cells. Activity of the mutated enhancer is observed in umbilical
cord (uc), intersomitic artery (isa) (A) and the head vasculature (C).
Sectional analysis shows lacZ expression in the endothelial cells of
head vasculature (inset in C), ductus venous of E12.5 (F, arrow) and E11.5
embryos (H, arrowhead in inset). Within the heart, aberrant enhancer activity
was observed in the endocardial cells of the trabeculated ventricular outlet
in E12.5 (E, arrowhead in the inset) and E11.5 hearts (G, marked by an
asterisk), and sinus venous valves (G, arrowhead). The dysregulated enhancer
activity was also observed in the cushion mesenchymal cells (E, inset).
Activity of the mutated enhancer appeared to be increased in the pro-valve
endocardial cells (B and D, arrowheads). (I) A table summarizes transient
transgenic experiments with the mutated constructs. TG, transgenic embryos;
EC, endocardial cell expression; ET, ectopic expression.
|
|
 |
Discussion
|
---|
The results of this study offer an initial elucidation of how
endocardial-specific gene expression can be achieved during valvulogenesis and
cardiac septation. We demonstrate that a sequence of 781 bp within the first
intron of the murine Nfatc1 functions as an autonomous cell-specific
enhancer to direct strong and highly specific expression in pro-valve
endocardium during cardiac development in vivo. Activation of this enhancer
element is only observed in the endocardial cells of the AVC and OFT. It is
not detected during initial differentiation of the endocardium from the
mesoderm in the E7.5 embryo, nor does it drive expression in the transformed
endocardial cells at the time when the endocardial cushions are forming and
subsequently remodeled into cardiac valves.
It has been shown that mesenchymal cushion formation results at least
partially from EMT within the heart tube as a consequence of interaction
between both localized myocardial cues and adjacent endocardial responsiveness
(Eisenberg and Markwald, 1995
;
Markwald et al., 1996
).
Tgfß/Bmp signaling pathways appear to modulate this
myocardial-endocardial interaction in both the avian and mouse embryos
(Boyer et al., 1999
;
Brown et al., 1999
). Yet, the
transcriptional circuitry that governs the phenotypic changes of these
specialized endocardial cells during EMT and later valvulogenesis has not been
extensively studied. The enhancer described in our studies could function as a
`genetic switch' that turns on and later turns off Nfatc1 expression in
response to signals elicited, presumably, from myocardium of the endocardial
cushions. Identification of this endocardial enhancer thus provides a novel
genetic marker for the unique pro-valve endocardial cells. It may also serve
as a genetic readout for the inducible myocardial cues allowing the nature of
such signals to be deduced.
Scanning of the 250 bp necessary endocardial enhancer sequence reveals
binding sites for several transcription factors, including Smad, Gata and
Nfat, which mediate signals known to be involved in regulating EMT and/or
later valve formation. We and others have shown an autoregulation of Nfatc1
expression in T cells in the adult animal through the Nfat sites in its P1
promoter (Chuvpilo et al.,
2002
; Zhou et al.,
2002
). The sustained high expression of nuclear activated Nfatc1
in endocardium during cardiac valve formation suggests that a similar
autoregulatory paradigm may also operate for Nfatc1 expression in the
developing endocardium. Consistent with this notion, there are five consensus
Nfat sites located in the 250 bp sequence necessary for the endocardial
enhancer activity in vivo, and four of them are nested in the two conserved
ECEs. We demonstrated, using a genetic approach, that Nfatc1 expression is
required for maintaining the activity of the endocardial enhancer. We also
determined, using EMSA and ChIP assays, that this Nfatc1-dependent enhancer
activity is probably the direct result of interaction of Nfatc1 with one or
more Nfat sites in the 250 bp sequence.
Tgfß/Bmp-Smad pathways play a prominent role during EMT. Both in vitro
and in vivo studies have indicated that ligands of Tgfß/Bmp receptors are
strong inducers or positive regulators of EMT, and are essential for later
morphogenesis of cardiac valves. However, the nuclear events or targets of
Tgf/Bmp activation in the endocardial cells are not fully characterized.
Recently, Gata transcription factors have emerged as another cohort of
important regulators of EMT. Disruption of Gata4 interaction with its
co-factor, Fog2, by a `knock-in' mutation (Gata4KI/KI)
(Crispino et al., 2001
) or
endothelial-specific deletion of Gata co-factor, Fog1, result in both OFT and
AVC defects (Katz et al.,
2003
). Furthermore, in vitro data suggest that an interaction
between Gata5 and Nfatc1 may be important for endocardial cell differentiation
(Nemer and Nemer, 2002
). In
our study, we found that binding sites of Gata and Smad factors in the
conserved enhancer region are not essential for the specificity of the
enhancer, although we could not rule out their effect on the level of enhancer
activity.
By contrast, the Hox site was found to be required for maintaining the
specificity of the enhancer by suppressing its activity outside the pro-valve
endocardial cells. Thus, the intact Hox-binding site represents a negative
cis-element where its interaction with its binding factors in non pro-valve
endocardial tissues is probably required for limiting Nfatc1 expression
outside of the pro-valve endocardial cells. Hox factors consist of a large
number of homeobox transcription proteins and the binding site for these
factors is relatively diversified, and thus less defined when compared with
the Nfat site. We do not know which Hox factor plays a role in suppression of
the enhancer activity outside the pro-valve endocardial cells, such as
endocardial cells of ventricular trabeculae and transformed endocardial cells.
In the developing heart, Msx1, previously known as Hox7, is expressed in the
developing endocardium (Lyons et al.,
1992
; Robert et al.,
1989
), while a closely related gene, Msx2, is highly expressed in
the cushion cells that are transformed from endocardial cells
(Abdelwahid et al., 2001
). In
addition, other homeobox genes, iroquois 5 and iroquois 6, are only expressed
in the E11.5 endocardial cells lining the trabeculated myocardium
(Christoffels et al., 2000
).
Whether any or all of these factors are upstream regulators of this enhancer
is currently under investigation.
In summary, mesenchymalization of the endocardial cushions by EMT, and the
later remodeling of the cushions into the mature valves and septa are crucial
morphogenic processes susceptible to environment and genetic alterations that
result in common congenital heart diseases. These processes must require the
coordinated regulation of several signaling pathways. Our data provide initial
in vivo identification and characterization of a crucial enhancer required for
cell-specific autoregulation of Nfatc1 expression in the pro-valve endocardial
cells as well as suppression of its expression in those non-valve endocardial
cells. This work should facilitate further delineation of how multiple signals
are integrated at the transcriptional level to orchestrate valvulogenesis and
should provide valuable in vivo models to specifically investigate gene
function in pro-valve endocardium required for normal cardiac valve
formation.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. M. A. Brown for providing the mouse Nfatc1.ß plasmid, and
Drs P. Robson and S. Brandt for critical reading and helpful discussion. This
work was supported by a Scientist Development Grant from American Heart
Association (B.Z.) and funding from the National Institute of Health
(H.S.B.).
 |
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