Kru ¨ppel-Like Factor 2 Is Required for Normal Mouse
Aditi R. Chiplunkar1, Tina K. Lung1, Yousef Alhashem1, Benjamin A. Koppenhaver1, Fadi N. Salloum2,
Rakesh C. Kukreja2, Jack L. Haar3, Joyce A. Lloyd1,4*
1Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia, United States of America, 2Department of Internal Medicine,
Virginia Commonwealth University, Richmond, Virginia, United States of America, 3Department of Anatomy and Neurobiology, Virginia Commonwealth University,
Richmond, Virginia, United States of America, 4Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, United States of America
Kru ¨ppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress,
such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death by
mouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental role
for KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF22/2 embryos die earlier, by E11.5. E9.5 FVB/N KLF22/2
hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normal
single layer. By E10.5, traditional and endothelial-specific FVB/N KLF22/2 AV cushions are hypocellular, suggesting that the
cells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF22/
2 hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number of
mesenchymal cells migrating from FVB/N KLF22/2 AV explants into a collagen matrix is reduced considerably compared to
wild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/N
KLF22/2 embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF22/2 hearts
have largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF22/2 embryos have a delay in the formation of
the atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucose
dehydrogenase (Ugdh), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and Ugdh promoters in
chromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. In conclusion, KLF22/2
heart phenotypes are genetic background-dependent. KLF2 plays a role in EMT through its regulation of important
Citation: Chiplunkar AR, Lung TK, Alhashem Y, Koppenhaver BA, Salloum FN, et al. (2013) Kru ¨ppel-Like Factor 2 Is Required for Normal Mouse Cardiac
Development. PLoS ONE 8(2): e54891. doi:10.1371/journal.pone.0054891
Editor: Elena Aikawa, Brigham and Women’s Hospital, Harvard Medical School, United States of America
Received July 5, 2012; Accepted December 18, 2012; Published February 14, 2013
Copyright: ? 2013 Chiplunkar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health (NIH R01 DK074694). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Congenital heart defects (CHDs) are a leading cause of infant
morbidity and mortality (reviewed in ). Valve and septal defects
account for the majority of CHDs. Mutations in transcription
factor genes, including Nkx2-5  and the cardiac T-box gene
TBX5 , are important for normal valve development in
humans. Mutations or variants in other transcription factor genes
are likely to be involved in valve defects.
During mouse heart development, localized swellings of the
endocardial layer arise at approximately embryonic day 9.5 (E9.5),
and form the endocardial cushions of the atrioventricular (AV)
canal and the outflow tract. The endocardial cushions are formed
by endothelial to mesenchymal transformation (EMT). During
EMT, AV endocardial cushion cells undergo hypertrophy, loss of
cell-cell contacts, lateral mobility, formation of mesenchymal-like
cell processes (filopodia), and migration into the cardiac jelly
(Reviewed in ). Without normal cardiac jelly, endothelial cells
fail to transform and to migrate, resulting in hypoplastic
endocardial cushions . Extensive remodeling and proliferation
of the endocardial cushions occurs to form the adult heart valves.
By E10.5, the right and left atria have divided . The AV
endocardial cushion region also plays an important role in
septation of the heart (Reviewed in ).
Kru ¨ppel-like factor 2 (KLF2) is a member of a family of zinc
finger-containing transcription factors [8–10]. In the E9.5 mouse
heart, KLF2 mRNA is highly expressed in the endocardial cells of
the AV cushion regions , where it is likely induced by high
shear stress . The role of KLF2 in cardiac development was
studied by Lee et al., in knockout (KO) mice in a mixed genetic
background including C57BL/6J. In KLF2 KO and KLF2
endothelial conditional KO embryos, a cardiac phenotype is
observed as early as E10.5. These mice have thinning of the
myocardium, high output heart failure, and die by E14.5. These
KLF22/2 embryos also have cardiac functional defects due to
loss of vessel tone, but no role for KLF2 in cushion formation was
noted . In two other KLF22/2 models, embryos in a mixed
genetic background die between E12.5 and E14.5, and exhibit
hemorrhaging [1,13]. The zebrafish gene, klf2a, a homolog of
KLF2 in mouse and man, is important in valve development.
Knockdown of klf2a causes thicker and less flexible valves and
PLOS ONE | www.plosone.org1 February 2013 | Volume 8 | Issue 2 | e54891
increased regurgitation in the developing heart, compared to WT
In tissue culture, KLF2 plays a role as a molecular transducer of
fluid shear forces, thus directly or indirectly regulating a number of
endothelial genes [15,16]. Recent findings suggest that KLF2 plays
an important role in endothelial barrier function in adult mice. It
positively regulates expression of the tight junction protein
occludin and modification of myosin light chain that is important
for the integrity of the endothelial layer and to avoid vascular
leakage . In adult mice, KLF2 positively regulates the
expression of vasoprotective genes and inhibits expression of
pro-inflammatory genes in endothelial cells and macrophages,
preventing atherosclerosis .
klf2a knockdown results in abnormal zebrafish heart valve
development, and KLF2 is expressed in the mouse endocardial
cushion region. This suggests that KLF2 may be important in
the early stages of mammalian valve development. In this work,
we show that FVB/N KLF22/2 hearts are hyperplastic with
respect to cells lining the AV canal, and hypoplastic with
respect to endocardial cushion mesenchymal cells. The data
suggests that KLF2 regulates EMT, and also atrial septation.
KLF2 activates multiple important cardiovascular development
genes, suggesting mechanisms for its roles in the embryonic
Materials and Methods
This study was approved by the Virginia Commonwealth
University Institutional Animal Care and Use Committee (VCU
IACUC). VCU IACUC Protocol Number: AM10347.
Generation of Knockout and Transgenic Mice
The traditional KLF2 KO mouse model was developed by
targeting the gene with the hypoxanthine phosphoribosyl-trans-
ferase (Hprt) gene . KLF2+/2 adults were mated with FVB/
N or C57BL/6 mice for at least 12 generations to obtain KLF2+/
2 animals in the FVB/N or C57BL/6 genetic background. These
animals were then mated to obtain KLF22/2 embryos. FVB/N
and C57BL/6 KLF2+/2 mice were mated to obtain KLF2+/2
animals in a 50% FVB/N and 50% C57BL/6 genetic background
(mixKLF2+/2). mixKLF2+/2 adults were mated to obtain mix
WT and mix KLF22/2 embryos.
Tie2-cre transgenic animals were purchased from Jackson
Research Laboratories (Bar Harbor, ME). Mice with a KLF2
allele surrounded by loxP sites (floxed allele or KLF2fl/+)were
kindly provided by Dr. Jerry Lingrel, University of Cincinnati and
were generated as previously described . These animals were
mated with FVB/N mice for at least 12 generations to obtain
Tie2-cre and KLF2fl/flanimals in an FVB/N genetic background.
FVB/N Tie2-cre and KLF2fl/flanimals were then mated with
each other to obtain Tie2-cre, KLF2fl/+which were consequently
mated with KLF2fl/flanimals to obtain Tie2-cre, KLF2fl/flanimals
in the FVB/N background. These animals are designated FVB/N
Light and Electron Microscopic Studies
Embryos were prepared for sectioning by fixing in 2%
paraformaldehyde (PFA) and 0.25% glutaraldehyde, and embed-
ded in eponate 12. Serial cross-sections of entire E9.5 and E10.5
embryos of 7 and 5 mm, respectively, were obtained using an LKB
2128 Ultramicrotome. Sections were stained with Toluidine Blue,
and images were made using an Olympus BX41 compound
microscope and Olympus DP71 digital camera. E9.5 endocardial
cell counts were obtained for the central section of the AV canal
plus a section 2 sections anterior and a section 2 sections posterior.
The ends of the AV canal were designated as 2 endothelial cells
beyond the point where the AV canal widens into the ventricle or
the atria. E10.5 AV cushion mesenchymal cell counts were
obtained for the central section of all sections with endocardial
cushion tissue associated with the AV canal. The counts were
expressed in cells/mm2. Image J 1.46 software was used to trace
the irregular outline of the cushion and measure the area. For
electron microscopy, embryos were sectioned at 100 nm with an
LKB 2128 Ultratome and stained with 5% Uranyl Acetate and
Reynold’s Lead Citrate. Images were taken on a JEOL JEM-1230
TEM with a Gatan Ultrascan 4000 digital camera.
Benzidine staining was performed on cryosections of 4% PFA-
fixed FVB/N WT and KLF22/2 embryos. Sections were
submerged in 1X PBS for 1 hour prior to benzidine staining as
described previously . Sections were then incubated in
methanol for 15 seconds, 1% benzidine in methanol for 5
minutes, 2.5% hydrogen peroxide in 70% ethanol for 3 minutes,
and washed with DI water for 2.5 minutes.
FVB/N KLF2+/2 mice were mated to generate E10.5 WT
and KLF22/2 embryos. Embryos were fixed in 4% PFA in
Millonig’s buffer, washed in 1X Phosphate Buffered Saline
Tween-20 (PBST), and incubated in 0.5% hydrogen peroxide,
0.5% normal goat serum (NGS) in PBST. The samples were
placed in antibody blocking solution (10% goat serum in PBST)
and incubated in primary antibody, PECAM (1:200) (BD
Biosciences). The embryos were incubated with secondary
antibody, biotinylated Anti-Rat IgG (1:500) (Abcam) and BD
Pharmingen Streptavidin-Horseradish Peroxidase (Sav-HRP) and
BD Pharmingen 3,39diaminobenzidine (DAB) chromogen in
H2O2buffer and fixed in 4% PFA. The embryos were frozen
and sectioned (10 mm) as described previously [21,22].
Alcian Blue Staining
E10.5 WT and KLF22/2 embryos were fixed in a 2% PFA
and 0.25% glutaraldehyde solution and cryo-embedded. Cross-
sections of 10 mm were cut using a vibratome (Ultrapro 5000).
Sections were stained with Alcian Blue (Sigma Aldrich) and
Nuclear Fast Red (Sigma Aldrich) and observed using an Olympus
BX41 microscope. Images were made using an Olympus DP71
RNA was prepared from E10.5 AV canals, and quantitative
RT-PCR (qRT-PCR) was performed as previously described .
Some of the primer sequences were as previously described by
others: hyaluronan synthase 2 or Has2 , Tbx5  Notch1
[25,26] and Gata4 . The primer sequences for these and the
other genes, PECAM1, Ugdh, Sox9 and Tgfb2, are given in Table
S1. Mouse cyclophilin A mRNA was used as an internal standard
for normalization. mRNA amounts were quantified using SYBR
Green or Taqman reagents (Applied Biosystems). For assays using
SYBR Green chemistry, dissociation curves were generated, and it
was verified that only one product was amplified. A standard curve
from pooled cDNA samples was included in each run to measure
the relative amounts of unknown samples. Statistical significance
was determined using the Student’s t-test.
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KLF2+/2 adults were mated to obtain E10.5 embryos. Non-
invasive in utero fetal ultrasound using a VisualSonics Vevo 770
System and 40 MHz mechanical transducer (VisualSonics) was
performed on 33 E10.5 embryos. Three pregnancies for the FVB/
N and for the mixed genetic backgrounds were examined. The
mice were anesthetized using pentobarbital (30 mg/kg; intraper-
itoneal). The embryos were numbered for genotyping based on
their position in utero, as described previously . Maternal
temperature was maintained using a heat lamp, if required. The
40 MHz VisualSonics RMV transducer gives an axial resolution
of 30 mm. For each embryo, blood flow parameters, including
heart rate, blood flow velocities and volumes were measured, as
previously described . Cardiac output was calculated as stroke
volume multiplied by heart rate and expressed as beats per minute.
2D imaging was used to view the 3 or 4 chamber heart and
measure left ventricle ejection fraction. To statistically compare
the values in WT and KLF22/2, the Student’s t-test was used.
AV Explant Collagen Assay
AV regions were dissected from E10.5 FVB/N KLF22/2 and
WT hearts and explants were placed on a collagen matrix as
previously described [23,28–30]. After a 72 hour incubation, the
explants were observed and photographed using an Olympus
IX70 inverted microscope with Hoffman Modulation Optics. The
cell counts were performed at a single plane of focus, at which the
vast majority of the cells could simultaneously be observed.
Chromatin Immunoprecipitation Assay
ChIP assays were performed essentially as described previously
, using a KLF2 polyclonal antibody generated using a
previously described construct , and a negative control
preimmune serum. Briefly, for each biological replicate, approx-
imately 36106cells from ,8–10 E10.5 FVB/N WT AV regions
were cross-linked with 1% formaldehyde for 10 min at room
temperature. Chromatin was sheared to approximately 500 bp
using a Bioruptor sonicator (Diagenode, Sparta, NJ). Chromatin
was precleared using protein G, precipitated with KLF2 or
preimmune antiserum, and cross-links were reversed. DNA was
purified and analyzed using quantitative PCR (qPCR) and SYBR
Green chemistry. Primer sequences for qPCR are indicated in
Table S2. Fold enrichment was calculated as 2‘(Ctinput2 Cttest) and
expressed relative to the preimmune serum control.
The Chi-square test, Student’s t-test or ANOVA were used for
statistical analyses, as indicated. Standard deviation was used to
measure deviation from the mean, for all experiments. For all of
the statistical tests, p values #0.05 were considered significant.
KLF22/2 Embryos in the FVB/N Genetic Background Die
Matings between FVB/N KLF2+/2 mice resulted in the
expected number of embryos of each genotype at E9.5 and E10.5.
Out of 28 embryos from four KLF2+/2 matings, no (zero)
KLF22/2 E11.5 embryos were obtained (expected frequen-
cy=7). Chi-square analysis was performed to compare the
observed and expected frequencies (Table 1), and it was
determined that the number of KLF22/2 embryos was
significantly less than expected (p=0.0044). FVB/N KLF22/2
embryos die by E11.5, sooner than KLF22/2embryos in a mixed
genetic background, which die by E14.5 [1,11,13]. This suggests
that modifier genes in FVB/N affect the KLF22/2 phenotype.
E9.5 FVB/N KLF22/2 Mice have an Accumulation of Cells
Lining the AV Canal
In order to investigate the reasons for the earlier embryonic
death of KLF22/2 mice in the FVB/N genetic background,
serial sections of entire embryos were collected to assess
morphological abnormalities. Using light microscopy, anterior to
posterior cross-sections of E9.5 FVB/N KLF22/2 embryos were
analyzed. Compared to WT littermates, these KLF22/2
embryos appear grossly normal, but at the cellular level there is
a dramatic difference in the AV endocardial cushions. In WT
embryos, the AV cushions are lined by a single layer of endothelial
cells, as expected (Fig. 1A and 1C, n=3). However in the FVB/N
KLF22/2 embryos, there is an increased number of cells lining
the AV canal region, and these cells form multiple disorganized
layers (Fig. 1B and 1D, n=3). To determine whether the number
of endothelial cells in these E9.5 WT and KLF22/2 atrioven-
tricular cushions is significantly different, cell counts were
performed (Fig. 1E). There are 2-fold more cells lining the AV
canal in KLF22/2 than in WT. The Student’s t-test indicates
that this is a significant difference with a p-value of 0.0078. AV
morphological defects were not reported in KLF22/2 hearts
from mice in a mixed genetic background [1,11,13].
FVB/N KLF22/2 Embryos have Abnormal Endocardial
The AV cushion region defects in E9.5 FVB/N KLF22/2
mice were studied at the subcellular level using transmission
electron microscopy (TEM). TEM reveals that the E9.5 FVB/N
WT (Fig. 2A) and the KLF22/2 (Fig. 2B) AV canals are patent
and contain erythroid cells. The KLF22/2 AV canal is not lined
by typical endocardial cells that are squamous (flat) but instead by
cells that are bulbous with numerous cytoplasmic processes
extending into the lumen of the AV canal (Fig. 2D). WT cells
are squamous and do not have projections or have projections
towards the cushions, rather than lumen, as expected (Fig. 2C).
Table 1. Number of embryos observed and expected from FVB/N KLF2+/2 matings.
embryos WT OWT E KLF2+ +/2 2 O KLF2+ +/2 2 EKLF22 2/2 2 O KLF22 2/2 2 E Chi-square p-value
E9.5 268 6.5 12 136 6.50.7939
E11.52712 (0)6.7515 (1)13.50 (2)6.75 0.0044*
Parentheses indicate number of dead embryos at E11.5. O is observed; E is expected. Asterisk indicates a statistically significant difference between O and E.
KLF2 Is Required in Mouse Cardiac Development
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Moreover, additional cells lie adjacent to the endocardial layer in
KLF22/2 compared to WT, which makes the cushions appear
disorganized and non-laminar.
E10.5 FVB/N KLF22/2 Embryos have Hypoplastic AV
Endocardial Cushions and Other Cardiac Abnormalities
To examine the KLF22/2 AV endocardial phenotype at a
later stage of development, light microscopy was performed at
E10.5 (n=3). The cardiac abnormalities in FVB/N KLF22/2
are more severe at E10.5 than at E9.5. The cells in the AV canal
region and endocardial cushions in FVB/N KLF22/2 (Fig. 3C
and 3D) are disorganized compared to somite matched FVB/N
WT controls (Fig. 3A and 3B). In E10.5 FVB/N KLF22/2
embryos, the AV cushions are hypocellular (Fig. 3D at *)
compared to FVB/N WT which are highly populated with
mesenchymal cells (Fig. 3B at *). In FVB/N KLF22/2, the
endocardial cells are evidently unable to transform into mesen-
chymal cells and migrate into the cushions, and therefore
endothelial-like squamous cells accumulate lining the AV canal.
Moreover, the E10.5 FVB/N KLF22/2 heart has only one
atrium (Fig. 3C), whereas somite-matched FVB/N WT hearts
have a left and right atrium at this time point (Fig. 3A), indicating
that there is also an atrial septal abnormality in the septum
primum in the mutants. Additionally, in the E10.5 FVB/N
KLF22/2 heart (Fig. 3C), the myocardium is thinner than in the
FVB/N WT heart (Fig. 3A), as previously reported . KLF2 is
not expressed in the myocardium, thus its effect on myocardial
development must be indirect.
KLF22/2) hearts show hypocellular endocardial cushions,
disorganized AV canal regions, and delayed atrial septal formation
(Fig. 3G and 3H), similar to the traditional KLF2 KO. Using this
model, the deletion of the KLF2 gene in the heart is quite
complete at approximately 84% (D. Vinjamur, unpublished data).
Negative control littermates having the floxed KLF2 gene without
Tie2-cre are unaffected, as expected and shown in Fig. 3E and 3F.
This suggests that KLF2 has an endothelial cell-autonomous role
in the AV cushion region.
The E10.5 FVB/N KLF22/2 heart morphological phenotype
is more severe than that previously reported by Lee et al. for mice
in a mixed genetic background, which had only myocardial
thinning . To confirm that the KLF22/2 phenotype varies in
different genetic backgrounds, matings were carried out to obtain
KLF22/2 embryos in a controlled genetic background that is
50% FVB/N and 50% C57BL/6 (mix KLF22/2). Light
microscopy studies on E10.5 mix KLF22/2 indicate that the
AV canal and cushion morphology in these embryos (Fig. 3K and
3L) is comparable to somite- and genetic background-matched
mix WT embryos (Fig. 3I and 3J). The endocardial cushions in
mix KLF22/2 embryos do not have a drastic reduction in
mesenchymal cells (Fig. 3L). However, mix KLF22/2 embryos
(Fig. 3K) have thinner myocardium than mix WT (Fig. 3I), as
previously reported . C57BL/6 KLF22/2 hearts (Fig. 3O
and 3P) have apparently normal AV cushions like mix KLF22/2
and C57BL/6 WT (Fig. 3M and 3N), and thinner myocardium
like mix KLF22/2 and FVB/N KLF22/2. C57BL/6 KLF22/
2 hearts (Fig. 3O) have atrial septal abnormality similar to that
observed in FVB/N KLF22/2 (Fig. 3C), but not found in mix
KLF22/2 (Fig. 3K). To quantify the mesenchymal cell
hypocellularity of the AV endocardial cushions, cell counts were
Figure 1. E9.5 FVB/N KLF22 2/2 2 atrioventricular cushions have accumulated cells lining the AV canal. (A) and (C) are WT (n=4) and (B)
and (D) are KLF22/2 (n=4) hearts. Micrographs A and B (magnification 50X) show atrial (At) and ventricular (V) chambers. The boxes or brackets
enclose the endocardial cushions (EC) shown at higher magnification in C and D (400X). Endo: Endocardial cells; Myo: Myocardium; Ery: erythroid cells;
Mes: Mesenchymal cells. (E) Bar chart representing the number of cells/mm2 lining the AV canal of E9.5 WT and KLF22/2 embryos. The Student’s t-
test indicates that the number of cells is significantly greater in KLF22/2 than in WT (p=0.0078). n=4.
KLF2 Is Required in Mouse Cardiac Development
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performed for E10.5 WT and KLF22/2 embryos in the FVB/N,
C57BL/6 and mixed genetic backgrounds, and for the FVB/N
Tie2-cre KLF22/2 (Fig. 3Q). As expected, FVB/N KLF22/2
and Tie2-cre KLF22/2 showed highly significant (p,0.001)
decreases in the number of mesenchymal cells in the endocardial
cushion tissue associated with the AV canal, compared to controls.
Interestingly, C57BL/6 KLF22/2 embryos also showed a
statistically significant (p=0.03) decrease in the mesenchymal cell
count, suggesting that there is some role for KLF2 in EMT in this
strain. There may be unique recessive modifier genes involved in
the decreased AV mesenchymal cell numbers in FVB/N and in
C57BL/6 KLF22/2 hearts, because the number is normal in
mix KLF22/2 hearts.
Unlike WT (Fig. 4A), in FVB/N KLF22/2 embryos, erythroid
cells are observed outside of the dorsal aortas, suggesting possible
hemorrhaging (Fig. 4B and 4C). Alternatively, the presence of
erythroid cells outside of the FVB/N KLF22/2 dorsal aortas
may be due to an inability of hematopoietic progenitors,
originating in the para-aortic mesenchyme, to reach the aortic
endothelium and enter circulation . To confirm that these cells
are erythroid, benzidine staining was performed. In FVB/N
KLF22/2 embryos, there are benzidine-positive cells in the tissue
surrounding the dorsal aorta (Fig. 4E), but they are found only
within the dorsal aorta in FVB/N WT (Fig.4D). These results were
replicated in E10.5 FVB/N WT and KLF22/2 hearts at the 34
and 36 somite stages. There are no blood cells in the tissues
surrounding the dorsal aortas in mix KLF22/2 embryos (Fig. 4G
and 4H), which look comparable to mix WT (Fig. 4F) and FVB/N
WT (Fig. 4A).
The outflow tract endocardial cushions in two out of three
E10.5 FVB/N KLF22/2 embryos examined had an abnormal,
non-laminar accumulation of endocardial cells lining the lumen.
These same embryos also had a smaller number of mesenchymal
cells in the outflow tract cushions than WT (Fig. S1). Therefore,
although the outflow tract phenotype is not completely penetrant,
there are similarities between the AV and outflow tract phenotypes
in FVB/N KLF22/2 mice.
Cells Accumulated in the FVB/N KLF22/2 AV Canal have
To further define the role of KLF2 in EMT, the cells
accumulating in the FVB/N KLF22/2 AV canal were studied.
Immunohistochemical staining was performed using the endo-
thelial specific PECAM (CD31) antibody. The cells accumulated
in the FVB/N KLF22/2 AV canal are CD31 positive,
indicating that they have endothelial characteristics (results from
two embryos shown in Fig. 5C–5F). The FVB/N WT positive
control has an organized layer of CD31 positive endothelial
cells lining the AV canal (Fig. 5A and 5B). A negative control,
Figure 2. Electron microscopy of E9.5 FVB/N KLF22 2/2 2 atrioventricular cushions shows abnormal endocardial cell morphology. In
WT (A), the endocardium is one cell-layer thick but in KLF22/2 (B), there are multiple disorganized cell layers. The boxes enclose the AV canal. At
higher magnification, endocardial cells in KLF2 KO (D) extend cytoplasmic projections or filopodia-like extensions towards the lumen of the AV canal,
which are not observed in WT (C). Arrowhead indicates abnormal cytoplasmic projection towards lumen in KLF22/2. *indicates non-laminar cells
accumulated at the lining of the AV canal that are observed in KLF22/2 but not in WT.
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Figure 3. E10.5 FVB/N but not mix and C57BL/6 KLF22 2/2 2 atrioventricular endocardial cushions are hypoplastic and disorganized.
(A) and (B) are micrographs of FVB/N WT heart; (C) and (D) are micrographs of FVB/N KLF22/2 heart. The light micrographs, A and C (magnification
100X), show the structure of the E10.5 heart including the atrial (At) and the ventricular chambers (V) and B and D show the endocardial cushion
regions that are in the boxes in A and C respectively, magnified at 200X. The red dashed lines indicate the positions of the hypoplastic FVB/N KLF22/
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Expression of PECAM1 mRNA was significantly higher in
FVB/N KLF22/2 than in FVB/N WT AV canals as shown
by quantitative reverse transcriptase-PCR (qRT-PCR, Fig. 5G).
This suggests that there is an increased number of cells
expressing PECAM1mRNA, and/or PECAM1 expression per
cell is increased in KLF22/2. However, because an accumu-
onlywith secondary antibody,had nostaining.
lation of endothelial-like squamous cells lining the AV canal is
observed in KLF22/2, there is a high probability that there
are a higher number of cells expressing PECAM1 mRNA.
These findings support the premise that there is abnormal EMT
in the AV cushions of FVB/N KLF22/2 embryos and that
the cells abnormally accumulating at the AV canal are
endothelial cells that are unable to transform.
2 AV cushions in D, compared to normal AV cushions in H and L. (G) and (H) are micrographs of Tie2-cre KLF22/2, and (E) and (F) are WT littermate
controls without Tie2-cre; all are in the FVB/N background. (I) and (J) are micrographs of mix WT; (K) and (L) are micrographs of mix KLF22/2 hearts.
(M) and (N) are micrographs of C57BL/6 WT; (O) and (P) are micrographs of C57BL/6 KLF22/2 hearts. (Q) Bar chart representing the number of
mesenchymal cells/mm2in the endocardial cushion tissue associated with the AV canal. Counts were performed of all cells in a single central section
from E10.5 WT or KLF22/2 hearts in all three genetic backgrounds and for Tie2-cre KLF22/2. Student’s t-test indicates that the number of
mesenchymal cells is decreased in FVB/N KLF22/2, Tie2-cre KLF22/2 and C57BL/6 KLF22/2, compared to WT. Mes: Mesenchymal cells; Endo:
Endothelial cells; Ery: Erythroid cells; Myo: Myocardium. n=3–5 hearts for histological staining, and n=3 hearts for mesenchymal cell counts. WT and
KO embryos for each background are somite matched. Asterisks* indicate AV endocardial cushion region. Arrowheads show atrial septum that is
forming in A, E, I, K and M; the atrial septum is absent in C, G and O.
Figure 4. E10.5 FVB/N KLF22 2/2 2 but not mix KLF22 2/2 2 embryos have erythroid cells outside of the dorsal aortas. (A) is a light
micrograph (100X) of the FVB/N WT E10.5 dorsal aortas (DA). Erythroid cells (Ery) are found only within the aortas. (B) FVB/N KLF22/2 have a number
of erythroid cells in tissue surrounding the DA. The box indicates the region that is magnified in C (200X). (C) Erythroid cells within and outside the
vessel in FVB/N KLF22/2. (D) and (E) are light micrographs of benzidine-stained FVB/N WT and KLF22/2 sections, respectively (400X). Ery indicates
benzidine-positive, brown-colored erythroid cells. Mes indicates mesenchymal cells. (F), (G) and (H) are mix WT (100X), mix KLF22/2 (100X) and mix
KLF22/2 (200X), respectively, showing normal aortas with erythroid cells within the vasculature only. (n=3 each).
KLF2 Is Required in Mouse Cardiac Development
PLOS ONE | www.plosone.org7 February 2013 | Volume 8 | Issue 2 | e54891
E10.5 FVB/N KLF22/2 Hearts have Reduced
Glycosaminoglycans in the Extracellular Matrix
The accumulation of endothelial-like cells in the AV canal and
the absence of mesenchymal cells in endocardial cushions in FVB/
N KLF22/2 mice suggests that KLF2 regulates EMT during AV
cushion formation. Cardiac jelly is a prerequisite for endothelial
cell transformation and migration. Glycosaminoglycans form the
major component of the cardiac jelly and are essential for
endocardial cushion EMT. Therefore, alcian blue was used to
stain glycosaminoglycans in WT and KLF22/2 embryos in the
FVB/N and mixed genetic backgrounds. At E10.5, FVB/N
KLF22/2 hearts have a vast reduction of glycosaminoglycans in
the cardiac jelly (Fig. 6B and 6D) compared to WT (Fig. 6A and
6C). This complements the data indicating that KLF2 is required
for EMT in the endocardial cushions. Alcian blue staining appears
normal in mix KLF22/2 hearts (Fig. S2), indicating that the
reduction of GAGs is specific to FVB/N KLF22/2. To examine
whether KLF2 ablation results in reduced expression of genes
Figure 5. E10.5 FVB/N KLF22 2/2 2 hearts have accumulated endothelial cells lining the AV canal. Immunohistochemical staining (n=2) was
performed using an endothelial cell specific mouse PECAM (CD31) antibody. A goat anti-mouse secondary antibody conjugated with HRP was used,
and reacted with diaminobenzidine (DAB) for detection. Brown coloration of cells indicates a CD31 positive cell type. (A) The cells lining the AV canal
in FVB/N WT are CD31 positive. (B) Higher magnification of WT AV region. (C) and (E) Cells accumulated in the AV canal in two different FVB/N
KLF22/2 hearts are CD31 positive, indicating that they are endothelial cells. (D) and (F) Higher magnifications of KLF22/2 AV regions show
accumulation of stained cells. Asterisks* indicate AV endothelial cushion region. A, C and E are 100X magnification; B, D and F are 200X. (G) qRT-PCR
shows a 2-fold increase in expression of PECAM mRNA in FVB/N KLF22/2 AV canals compared to FVB/N WT (p=0.002, n=7). The amount of PECAM
mRNA in WT was designated 100%.
KLF2 Is Required in Mouse Cardiac Development
PLOS ONE | www.plosone.org8 February 2013 | Volume 8 | Issue 2 | e54891
encoding enzymes involved in the synthesis of glycosaminoglycans,
quantitative RT-PCR was performed using RNA from WT and
KLF22/2 AV canals. As shown in Fig. 6E, the expression of
UDP-glucose dehydrogenase (Ugdh) mRNA was significantly
reduced in FVB/N KLF22/2 compared to FVB/N WT, but
similar amounts were expressed in mix WT and mix KLF22/2.
This correlates with the reduced alcian blue staining in FVB/N
KLF22/2, but not in mix KLF22/2, compared to WT. UGDH
converts UDP-glucose to UDP-glucuronic acid, which is required
for the biosynthesis of GAG components like hyaluronan, heparin
sulfate and chondroitin . Expression of the hyaluronan
synthase 2 (Has2) gene was not reduced in FVB/N KLF22/2
compared to WT AV canals (data not shown). Therefore a
reduction in Has2 is not likely to be the cause of the reduced
GAGs observed in FVB/N KLF22/2 hearts.
A Reduced Number of Mesenchymal Cells Invade the
Collagen Gel in FVB/N KLF22/2 AV Explant Assays
To determine whether the EMT defect is caused solely by a
defect in the cardiac jelly, or also by an abnormality in the
endocardial cells in FVB/N KLF22/2 hearts, E10.5 AV explant
assays were performed using a collagen gel. FVB/N WT explants
underwent EMT and the mesenchymal cells migrated into the
collagen matrix during the 72 hour incubation (Fig. 6F). Com-
pared to WT explants, KLF22/2 explants showed a significant
reduction in the number of mesenchymal cells that migrated into
the collagen matrix (Fig. 6G and 6H). The KLF22/2 explants
have at least a 10-fold reduction in the number of transformed
cells compared to WT (Fig. 6H). In addition, the fraction of
migrated cells that are transformed is greater than activated cells in
WT explants, but the reverse is true in KLF22/2. This suggests
that a defect in the endocardial cells, as well as in the cardiac jelly
composition, is responsible for abnormal EMT in the FVB/N
KLF22/2 AV cushions.
FVB/N KLF22/2 Hearts Exhibit Abnormal Cardiac
Echocardiography was performed on E10.5 FVB/N WT, FVB/
N KLF22/2, mix WT and mix KLF22/2 embryos. The heart
rate was not significantly different in embryos of the 4 genotypes
(Fig. S3A), but cardiac output and ejection fraction are signifi-
cantly higher in FVB/N KLF22/2 than in WT embryos (Fig.
S3B and S3C left side, respectively). The descending aorta velocity
in FVB/N KLF22/2 hearts is significantly lower than WT (Fig.
S3D left side). Higher than normal cardiac output and ejection
fraction were observed in E11.5 KLF22/2 embryos in a mixed
genetic background , but these features were not evident in
E10.5 mix KLF22/2 (Figs. S3B, S3C and S3D; right side). It is
difficult to reconcile the specific abnormalities in the heart
parameters in FVB/N KLF22/2 with the observed morpholog-
Cardiovascular Genes Important for AV Cushion
Development and Septation are Downregulated in FVB/
N KLF22/2 Compared to WT AV Regions
KLF2 is a transcription factor expressed in endothelial cells. To
begin to elucidate the molecular mechanism by which KLF2
ablation results in heart development and septation abnormalities,
the amounts of expression of candidate genes important in AV
cushion development (Tbx5, Sox9, Tgfb2, Notch1, Gata4) and
atrial septation (Tbx5, Gata4) (Reviewed in ) were quantified in
AV regions dissected from FVB/N WT and KLF22/2 hearts.
Each of these genes has at least one consensus KLF2 binding site
in its promoter (CCACCC and CCGCCC) [10,32], within 500 bp
upstream of the transcription start site (Table S3).
The mRNAs for three cardiovascular transcription factors,
Tbx5, Gata4 and Sox 9, showed significantly reduced expression
in FVB/N KLF22/2 compared to WT AV regions (Figs. 7A–
7C), indicating that these genes are, directly or indirectly,
positively regulated by KLF2. FVB/N Tie2-cre KLF22/2 AV
canals show a similar and significant reduction in the expression of
the Tbx5, Gata4 and Sox9 genes, compared to controls without
Tie2-cre (Fig. S4). Interestingly, expression of these genes is not
reduced in mix KLF22/2 compared to mix WT, consistent with
the absence of the AV cushion and atrial septal phenotypes in mix
KLF22/2. Apparently, strain-specific modifier genes differen-
tially affect the expression of Ugdh, Sox9, Tbx5 and Gata4 mRNA
in response to KLF2 ablation. Other roles of Gata4 and Tbx5 are
discussed in detail later, but Gata4 and Tbx5 double heterozygous
knockout mice show myocardial thinning [24,35,36], like
KLF22/2. Mix KLF22/2 has myocardial thinning but no
reduction of Gata4 and Tbx5 mRNA, indicating that an
additional unknown gene(s) is related to this phenotype. Sox9
plays an important role during endocardial cushion EMT as well
as valve remodeling . The Notch1 and Tgfb2 genes are
important for atrioventricular development, but these mRNAs are
not expressed significantly differently in KLF22/2 and WT
hearts, whether the animals are in an FVB/N or a mixed genetic
background (data not shown). The data suggest that Tbx5, Gata4,
Sox9 and Ugdh are downstream of KLF2 and have a genetic
background specific role in AV endocardial cushion EMT, atrial
septation and cardiac jelly synthesis. More than one cardiovascular
gene shows reduced expression by 2–3 fold in the absence of
KLF2, suggesting that the KO phenotype is an outcome of
dysregulation of multiple downstream targets of KLF2.
KLF2 Binds to the Mouse Gata4, Tbx5 and Ugdh
KLF2 positively regulates mRNA expression of Gata4, Tbx5,
Sox9 and Ugdh. To better understand the mechanism of KLF2
regulation, chromatin immunoprecipitation (ChIP) assays using a
KLF2 polyclonal antibody  were performed using cells from
E10.5 FVB/N WT mouse AV regions. Due to the limited
availability of tissue per sample, between eight and ten E10.5 AV
regions were pooled for each ChIP assay. The Gata4, Tbx5 and
Sox9 promoters have multiple potential KLF2 binding sites, and
the Ugdh promoter has a single site (Table S3). Due to assay
limitations, the two Tbx5 and two Sox9 binding sites could not be
distinguished from each other, and were tested simultaneously.
Two regions of the Gata4 promoter, each containing two putative
KLF2 binding sites (designated 2100 and 2411), were tested for
KLF2 enrichment. Quantitative PCR (qPCR) was used to
determine the fold-enrichment of KLF2 at each promoter region,
by comparing ChIP assays with KLF2-antiserum to pre-immune
serum. The data in Figure 7D indicate that KLF2 showed an
approximately 70-fold enrichment at the Tbx5 promoter, 25-fold
enrichment at the Ugdh promoter, and 15-fold enrichment at the
proximal Gata4 promoter site (2100) compared to negative
control assays using pre-immune serum. No significant KLF2
enrichment was observed at the Sox9 promoter. As a negative
control, KLF2 does not bind to the promoter of the b-actin gene.
The ChIP assays thus indicate that KLF2 binds to the Gata4,
Tbx5 and Ugdh promoters, and therefore may directly regulate
these genes. No evidence was obtained to indicate that KLF2
directly regulates the Sox9 gene.
KLF2 Is Required in Mouse Cardiac Development
PLOS ONE | www.plosone.org9February 2013 | Volume 8 | Issue 2 | e54891
Cardiovascular development and morphogenesis is a complex
process, involving a number of highly conserved transcription
factors and signaling pathways . KLF2 plays a multi-faceted
role in cardiovascular development. It is expressed in the
endocardium of the developing heart. The accumulation of
endothelial-like cells lining the AV canal, reduced EMT, delayed
atrial septal formation, and the absence of normal cardiac jelly
composition are novel phenotypes for FVB/N KLF22/2 mice,
and therefore may be related to the earlier embryonic death in the
FVB/N genetic background. Our studies also identify putative
effectors downstream of KLF2 that may impact each of these
processes in the embryonic heart.
KLF2 is important for vascular integrity . Kuo et al.  and
Wani et al.  reported hemorrhaging in the abdominal and
cardiac outflow tract region in KLF22/2 embryos. To the
contrary, no hemorrhaging was observed in the KLF22/2
embryos examined by Lee et al. . This discrepancy may be
partially explained by the current study. Erythroid cells were
observed outside E10.5 KLF22/2 dorsal aortas in FVB/N but
not in mixed genetic background embryos, indicating that this
phenotype is genetic background-specific. The genetic background
of the KLF22/2 mice used in the previous studies was not well
defined. It is theoretically possible that the dorsal aorta defect in
Figure 6. E10.5 FVB/N KLF22 2/2 2 hearts have abnormal cardiac jelly, and a reduction in transformed mesenchymal cells. Alcian blue
staining for extracellular matrix and counterstain with nuclear fast red was performed on cross-sections of E10.5 FVB/N WT and KLF22/2 hearts
(n=3). (A) and (C) are WT AV cushions; the nuclei are stained red and the extracellular matrix is stained blue (100X and 200X magnifications,
respectively). (B) and (D) are FVB/N KLF22/2 AV cushions with decreased blue staining, indicating reduced glycosaminoglycans (100X and 200X
magnifications, respectively). At: Atrium; V: Ventricle. Boxes indicate the AV endocardial cushion region. (E) qRT-PCR indicates a 2-fold decrease in
expression of Ugdh mRNA (p=0.0204) in FVB/N KLF22/2 but not mix KLF22/2 compared to WT AV region (n=7). (F) and (G) AV canal explants
were incubated in vitro on a collagen matrix for 72 hours, and the cells migrating into the matrix were observed (100X magnification). (F) FVB/N WT
explants show mesenchymal cells migrating into the collagen matrix (n=5). Arrows indicate mesenchymal cells. Round cells are activated but not
transformed. Stellate cells are activated and transformed into mesenchymal cells. (G) FVB/N KLF22/2 explants have less mesenchymal cells
migrating into the collagen matrix than WT (n=5), indicating an EMT defect in the FVB/N KLF22/2 endocardial cells. (H) The bar chart indicates
percentage of transformed cells in FVB/N WT and FVB/N KLF22/2 (n=3). The number of cells in WT is designated as 100%. KLF22/2 explants have a
greater than 10-fold decrease in mesenchymal cells in the collagen matrix compared to WT (p,0.001).
KLF2 Is Required in Mouse Cardiac Development
PLOS ONE | www.plosone.org10 February 2013 | Volume 8 | Issue 2 | e54891
FVB/N KLF22/2 embryos causes shear stress, resulting in the
AV cushion defect. However, this hypothesis is not favored
because the AV cushion defect is observed by E9.5, whereas the
dorsal aorta defect is first apparent at E10.5.
The importance of genetic background in cardiac development
has been demonstrated in a number of studies. Sakata et al.
studied Hey2 deficient mice and observed a spectrum of
cardiovascular anomalies that varied in the BALB/c and
C57BL/6 genetic backgrounds . Astrof et al. studied the role
of fibronectin in heart development; a null mutation in the gene
results in arrested heart development earlier in 129S4 than in
C57BL/6 embryos . The current study shows that the role of
KLF2 in the morphology and function of the developing heart is
also genetic background specific. In the FVB/N background, loss
of KLF2 results in an EMT defect in the AV cushion region,
delayed formation of the atrial septum, myocardial thinning and
death by E10.5. In the C57BL/6 background, KLF22/2 shows
delayed atrial septation and myocardial thinning. In a mixed
background the major defect in KLF22/2 hearts is myocardial
In this work, we have demonstrated that KLF2 binds the
promoters of, and positively regulates, the Tbx5 and Gata4 genes
in the mouse E10.5 AV region. An endocardial specific Gata4 KO
has multiple layers of endocardium in the AV canal, and
hypocellular AV cushions at E10.5 , similar to FVB/N
KLF22/2. Tbx5 KO embryos have hypoplastic endocardial
cushions , like FVB/N KLF22/2. These phenotypes
correlate with our observation that Gata4 and Tbx5 expression
is reduced 3-fold in the absence of KLF2 in FVB/N AV canals.
Interestingly, the Tbx5 and Gata4 proteins physically interact
during cardiac development. A heterozygous mutation (mG295S)
in the Gata4 gene disrupts these protein interactions, resulting in
cardiac defects like atrial septal defects (ASD), AV septal defects
(AVSD) and myocardial thinning beginning at E11.5 [35,36]. The
AVSD and ASD in these mice are known to result from abnormal
EMT and remodeling of endocardial cushions. Gata4 mG295S is
Figure 7. Cardiovascular genes are dysregulated in FVB/N KLF22 2/2 2 AV region. Quantitative RT-PCR (qRT-PCR) of AV region RNA was used
to test the amount of expression of genes important for AV cushion development (Tbx5 and Sox9) and atrial septation (Tbx5 and Gata4). Cyclophilin
A mRNA was used as a normalization control. FVB/N WT and mix WT were designated as100%, and KLF22/2 were compared to WT. (A–C) In E10.5
FVB/N KLF22/2 AV region there is significantly decreased expression of (A) Tbx5 (p=0.0175), (B) Gata4 (p=0.0164) and (C) Sox9 (p=0.019) mRNA
compared to FVB/N WT, but Mix WT and Mix KLF22/2 hearts have no differences in expression of these genes. Error bars indicate standard deviation.
n=5. D) ChIP assays were performed on cells obtained from E10.5 WT AV regions. Approximately 8 to 10 WT AV regions were pooled to obtain cells
for one replicate. Polyclonal anti-KLF2 and non-specific control pre-immune serum was used. The y-axis represents the relative fold-enrichment. The
mean pre-immune enrichment was designated as ‘1.0’ and the enrichment with KLF2 antisera was scaled appropriately. The x-axis indicates the
location of the primers used for qPCR; all were in gene promoters and are described in Table S2. Pr: Promoter. Primers specific for the b-actin gene
were used as a negative control. n=7 biological replicates.
KLF2 Is Required in Mouse Cardiac Development
PLOS ONE | www.plosone.org 11February 2013 | Volume 8 | Issue 2 | e54891
a missense mutation resulting in diminished DNA binding affinity
and transcriptional activity, making it similar to a null allele. In
mice with this Gata4 mutation and a null allele for Tbx5, Gata4+/
2Tbx5+/2, there is normal EMT but defective remodeling,
resulting in septal defects . Like these double heterozygotes,
FVB/N KLF22/2 embryos have about 50% less Gata4 and
Tbx5 mRNA than normal, and similarly remodeling is affected.
However, EMT is affected, indicating that the KLF22/2
embryos are more severely affected than Gata4+/2Tbx5+/2,
likely because KLF2 also controls other cardiac genes.
KLF2 binds to and positively regulates the UDP-glucose
dehydrogenase (Ugdh) gene. UGDH is expressed in the endocar-
dium and catalyzes conversion of UDP-Glucose to UDP-
Glucuronic acid (UDP-GA) . UDP-GA is further converted
to hyaluronic acid and other glycosaminoglycans by hyaluronan
synthase 2 (Has2) . There is an approximately 2-fold reduction
in Ugdh mRNA in the FVB/N KLF22/2 AV canal. This may
result in decreased production of UDP-GA and consequently
reduced glycosaminoglycans in the cardiac jelly. Interestingly, two
missense mutations in the Ugdh gene were recently identified in 3
patients with congenital valve defects . These mutations result
in structural defects in UGDH that significantly compromise
enzyme function . These findings support our hypothesis that
reduced UGDH contributes to the cushion defects in KLF22/2
Sox9 is a cardiovascular transcription factor expressed in
endothelial and mesenchymal cells in the endocardial cushion
region . In our study, the Sox9 gene is positively controlled by
KLF2, but the lack of evidence for KLF2 promoter binding
suggests that the regulation is indirect, or controlled by a more
distant DNA element. Sox9 KO results in hypoplastic endocardial
cushions and abnormal valve formation . A study by Lincoln
et al. showed that ablation of Sox9 in endocardial cells results in
reduced EMT . These phenotypes are similar to FVB/N
KLF22/2. The reduced expression of Sox9 in KLF22/2 hearts
could be attributed to a reduced number of mesenchymal cells in
the AV cushion. However, this seems unlikely because Has2
mRNA, which is also expressed in endocardial and mesenchymal
cells, does not show reduced expression in KLF22/2 AV regions.
The current study indicates that KLF2 plays an important role
in the synthesis of cardiac jelly, AV endocardial cushion EMT and
atrial septation, by regulating several important cardiac genes.
These genes include but are probably not limited to Ugdh (cardiac
jelly), Sox9 (EMT), and Tbx5 and Gata4 (EMT, AV cushion
development, and septation). KLF2 influences the development of
the AV cushions and atrial septum, and is likely required for
normal cardiac function. Future studies of KLF2 variants may be
relevant to the better understanding of human heart defects.
FVB/N WT outflow tract (OFT, 100X magnification) shows
mesenchymal cells in the outflow tract endocardial cushion region.
B) FVB/N KLF22/2 OFT (100X magnification) shows that the
endocardial cushions are hypocellular with respect to mesenchy-
mal cells. Mes: Mesenchymal cells; EC: endocardial cushion. Myo:
Myocardium. Embryos are somite matched (36 somites). Arrow-
head indicates non-laminar endothelial layer in FVB/N KLF22/
Light micrographs of E10.5 outflow tracts. A)
alcian blue. Alcian blue staining for extracellular matrix and
counterstain with nuclear fast red on cross-sections of mix WT and
mix KLF22/2 hearts (n=3, 200X magnification). A) mix WT
and B) mix KLF22/2 show alcian blue positive AV cushions
suggesting normal composition of GAGs in extracellular matrix.
At: Atrium; V: Ventricle.
E10.5 mix KLF22 2/2 2 AV cushions stain with
creased cardiac output, ejection fraction, and reduced
descending aorta velocity. (A) The heart rate is comparable in
all 4 genotypes. (B) Cardiac output and (C) ejection fraction are
higher in FVB/N KLF22/2 than FVB/N WT embryos
(p=0.005 and p=0.037, respectively), there is no difference
between Mix WT and Mix KLF22/2. (D) Descending aorta
velocity (DAvel) in FVB/N KLF22/2 hearts is significantly lower
than WT (p=0.01). Error bars indicate standard deviation. n=5.
E10.5 FVB/N KLF22 2/2 2 embryos have in-
creased expression of cardiovascular genes, similar to
the traditional KLF2 KO. Quantitative RT-PCR (qRT-PCR)
of AV region RNA was used to test the amount of expression of
genes important for AV cushion development (Tbx5 and Sox9),
cardiac jelly synthesis (Ugdh) and atrial septation (Tbx5 and
Gata4). Cyclophilin A mRNA was used as a normalization
control. WT (KLF2fl/fl, without Tie2-cre) were designated as
100% and Tie2-cre KLF22/2 was scaled appropriately. In E10.5
Tie2-cre KLF22/2 AV regions there is decreased expression of
Gata4 (p=0.0072), Tbx5 (p=0.006), Ugdh (p=0.0012) and Sox9
(p=0.0014) mRNA compared to WT. All animals are in an FVB/
N genetic background. Error bars indicate standard deviation.
Students’ t-test was used to compare WT and KLF22/2 gene
expression. The brackets indicate significant differences in mRNA
expression between WT and KLF22/2. n=6.
Tie2-cre KLF22 2/2 2 AV regions have de-
qRT-PCR primer sequences.
binding sites in the Tbx5, Gata4, Sox9 and UGDH
Location and sequence of the potential KLF2
ChIP primer sequences.
We are thankful to Dr. Jerry Lingrel for KLF2 KO and KLF2fl/+mice, Dr.
Stephen Sawyer for frequent and invaluable advice, Ms. Susan Walker and
Ms. Judy Williamson for technical advice on microscopy experiments, and
Ms. ShaCoria Winston for help in performing qRT-PCR experiments.
Divya Vinjamur supplied unpublished data. We thank Drs. Luca Brunelli,
Joey Barnett and Chris Brown for critically evaluating this manuscript.
Conceived and designed the experiments: ARC TKL BAK FNS JLH JAL
YA. Performed the experiments: ARC TKL BAK JLH. Analyzed the data:
ARC TKL BAK FNS RK JLH JAL YA. Contributed reagents/materials/
analysis tools: FNS RK JLH JAL YA. Wrote the paper: ARC BAK JAL.
KLF2 Is Required in Mouse Cardiac Development
PLOS ONE | www.plosone.org12February 2013 | Volume 8 | Issue 2 | e54891
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KLF2 Is Required in Mouse Cardiac Development
PLOS ONE | www.plosone.org13February 2013 | Volume 8 | Issue 2 | e54891