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First published online 18 October 2006
doi: 10.1242/dev.02597
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1 Division of Pediatric Cardiology, Department of Pediatrics, Vanderbilt
Children's Hospital
2 Department of Cell and Developmental Biology, Vanderbilt University Medical
Center, Nashville, TN 37232, USA.
3 Division of Genetic and Translational Medicine, Department of Genetics, The
University of Alabama at Birmingham, Kaul 768, 720 20th Street S., Birmingham,
AL 35294, USA.
4 Department of Pharmacology, Vanderbilt University Medical Center, Nashville,
TN 37232, USA.
5 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville,
TN 37232, USA.
* Author for correspondence (e-mail: scott.baldwin{at}Vanderbilt.edu)
Accepted 30 August 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Tgfß, Cardiogenesis, AV remodeling, DILV, Congenital heart disease, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hoffman and Kaplan, 2002). The
most common disorders are caused by malformations in septation and
valvulogenesis. Epithelium-mesenchyme transformation (EMT) in the
atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart is a
crucial process regulating initial valve formation, in which a subpopulation
of endocardial cells invade the extracellular matrix as the result of regional
interaction between the myocardium and endocardium
(Barnett and Desgrosellier,
2003). These mesenchymalized cushions serve as the primordia of
valves and septa, and develop into mature structures through complicated
remodeling processes. Proper cushion formation is required for simultaneous
and subsequent events in cardiac development, including chamber formation and
septation. Impairment of cushion development can cause various forms of CHD,
such as misalignment between atria and ventricles, and yet the underlying
molecular and cellular machineries are poorly understood.
The Tgfß family of cytokines play crucial roles in many biological
processes, including cardiovascular development
(Azhar et al., 2003;
Barnett and Desgrosellier,
2003). Tgfß signaling is initiated when homodimers of ligands
(Tgfß1, Tgfß2, Tgfß3) bind to and bring together the type-I and
type-II receptor kinases on cell membranes, and the ligand/receptor complex
subsequently activates downstream target genes
(de Caestecker, 2004;
Shi and Massague, 2003). Three
type I receptors (Alk1, Alk2, Alk5) and a single type II receptor (Tgfbr2)
have been found to mediate Tgfß signaling. Tgfbr2 can bind only Tgfß
ligands, and not other Tgfß superfamily members
(de Caestecker, 2004;
Shi and Massague, 2003). In
addition, Tgfbr3 (previously known as betaglycan, TßRIII) and endoglin
function as co-receptors in concert with Tgfbr2 to regulate Tgfß
ligand/receptor interaction (Barnett and
Desgrosellier, 2003). Thus, inactivation of Tgfbr2
eliminates known Tgfß signaling by inhibiting ligand/receptor interaction
(Bhowmick et al., 2004) without
directly affecting other signaling pathways.
Crucial functions of Tgfß ligands during EMT have been suggested from
previous studies using a 3D in vitro collagen gel system. In these AV explant
cultures, Tgfß ligands can substitute for the overlying myocardium to
activate EMT (Nakajima et al.,
2000; Potts and Runyan,
1989; Ramsdell and Markwald,
1997), and inhibition of Tgfß signaling with an antisense
oligonucleotide complementary to Tgfb3 mRNA, or with neutralizing
antiserums against Tgfß ligands or receptors, blocks EMT
(Boyer et al., 1999;
Brown et al., 1996;
Brown et al., 1999;
Camenisch et al., 2002).
However, the central role of Tgfß signaling during EMT has not been
supported by mouse genetic studies. No obvious valvular defect has been
observed in Tgfb1-/-
(Dickson et al., 1995),
Tgfb3-/- (Kaartinen et
al., 1995) or Tgfbr3-/- mice
(Stenvers et al., 2003).
Tgfb2-/- mice display a range of cardiac defects with
partial penetrance, and the valvuloseptal defects of these mice were thought
to be caused by abnormal cushion morphogenesis at later stages
(Bartram et al., 2001;
Sanford et al., 1997). The
discrepancy between in vitro and in vivo data may result from the
complementation by the remaining Tgfß ligands. To circumvent the
functional redundancy of Tgfß ligands and the early lethality of
Tgfbr2-/- mice (Oshima
et al., 1996), and to identify the primary cardiac cell type that
is responsive to Tgfß signaling, we applied a Cre/loxp system to
specifically inactivate Tgfbr2 in the myocardium or endothelium of
developing mouse hearts.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were crossed with
Tgfbr2loxp/loxp mice
(Bhowmick et al., 2004;
Chytil et al., 2002) to
acquire Tie2cre;Tgfbr2loxp/+ and
cTnTcre;Tgfbr2loxp/+ mice, respectively.
Tgfbr2loxp/loxp mice were crossed with Rosa26R
(R26R) mice (Soriano,
1999) (the Jackson Laboratory) to acquire
Tgfbr2loxp/+;R26R mice, which were then intercrossed to
generate Tgfbr2loxp/loxp;R26R/R26R mice. In performing
conditional gene inactivation experiments, we always used male
Tie2cre;Tgfbr2loxp/+ or male
cTnTcre;Tgfbr2loxp/+ mice to cross with female
Tgfbr2loxp/loxp;R26R/R26R mice. Embryo dissection,
sectioning and X-gal staining were performed as described previously
(Jiao et al., 2003;
Jiao et al., 2002).
Laser microdissection and PCR analysis
Frozen sections of E10.5 embryos (15 µm) were dehydrated and AV
endothelial/mesenchymal cells isolated using a PixCell II Laser Capture
Microdissection Scope (Arcturus), following the manufacturer's instructions.
Genomic DNA or RNA was isolated from the laser-captured cells using either the
PicoPure DNA Extraction Kit (Arcturus) or the PicoPure RNA Isolation Kit
(Arcturus). The RiboAmp HS RNA Amplification Kit (Arcturus) was used to
perform two rounds of linear amplification of mRNA on the isolated total RNA
samples. The primers used to amplify the recombined and unrecombined
Tgfbr2loxp allele, and for reverse transcriptase (RT)-PCR
analysis of Tgfbr2 mRNA, have been described previously
(Bhowmick et al., 2004).
Quantitative real-time PCR analysis was performed using the LightCycler-DNA
Master SYBR Green I Kit on a LightCycler Instrument (Roche). Semi-quantitative
RT-PCR analysis was performed using the OneStep RT-PCR Kit (Qiagen).
In vitro collagen gel assay
In vitro collagen gel assays were performed as described previously
(Camenisch et al., 2002).
Measurement of sizes of ventricles and AV cushions
The procedure for quantification of ventricular and cushion volumes was
essentially as previously described
(Kubalak et al., 2002).
Briefly, E12.5 embryonic hearts were sectioned in the frontal plane at 8.0
µm intervals and photomicrographs of all sections were taken with a digital
camera (RTSlider 2.3.0, Diagnostic Instruments) under identical conditions.
The sizes of the ventricular chambers and AV cushions of each section were
measured in artificial units using the software MetaMorph 5.0 (Universal
Imaging), following the manufacturer's instructions, and the sum of all
sections for each structure was then calculated as its `raw size'. The
relative cushion size was determined by dividing the raw size of the inferior
(or superior) cushion with the raw size of the left ventricle (LV) to minimize
the effect of variations in developmental stages among embryos. The relative
cushion size of control embryos was artificially set as 100%. The raw size of
the right ventricle (RV) was divided by the raw size of the LV to acquire the
ratio of RV/LV size. In total, sections from four control and four mutant
embryos were measured.
Immunofluorescent studies and in situ hybridization analysis
Immunofluorescent studies were performed as described previously
(Jiao et al., 2003). The
primary antibodies used in this study included a cyclin D1 monoclonal antibody
(BD Biosciences), an anti-Ki67 antibody (Novocastra), a Pecam antibody (BD
Biosciences) and an 
-smooth-muscle actin antibody (Sigma). Cy2
conjugated secondary antibodies were used for visualization. Samples were
examined with a laser-scanning confocal image system (Zeiss LSM510).
Quantification of the intensity of the fluorescence was performed using
MetaMorph 5.0. Section in situ hybridization analysis was performed as
described previously (Jiao et al.,
2003), using a probe corresponding to the first four exons of
Tgfbr2.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sanford et al., 1997), some of
which are likely to be caused by the disturbance of cardiac looping
(Bartram et al., 2001),
suggesting potential roles for Tgfß signaling in the myocardium. To study
myocardial-specific attenuation of Tgfß signaling, we inactivated
Tgfbr2 by crossing cTnTcre;Tgfbr2loxp/+ mice with
Tgfbr2loxp/loxp mice
(Chytil et al., 2002).
cTnTcre induces recombination early in the cardiomyocyte lineage
(E7.5), and efficiently inactivates target genes between E9.5 and E10.5
(Jiao et al., 2003)
(Fig. 1A,B). The
myocardium-specific deletion of Tgfbr2 was confirmed by PCR analysis
using genomic DNA isolated from myocardial cells or endocardial cells
(Fig. 1C,D). The recombined
allele could be detected only from myocardial cells and not from endocardial
cells, whereas the unrecombined allele was hardly detected from myocardial
cells.
The cTnTcre;Tgfbr2loxp/loxp animals were
isolated with an expected Mendelian ratio (
25%) at all stages examined
(Table 1), excluding embryonic
lethality of these animals. Two out of 24 mutant animals displayed cardiac
defects, with one embryo (E17.5) showing VSD (data not shown), and the other
(E16.5) showing VSD and DORV (Fig.
1E-H). The low incidence (8.3%) of cardiac defects in
cTnTcre;Tgfbr2loxp/loxp animals suggests that
myocardial Tgfß signaling is not essential for normal cardiogenesis.
However, perturbation of myocardial Tgfß signaling may cause embryonic
hearts to be more susceptible to deleterious genetic and/or environmental
factors, and may thus contribute to congenital abnormalities.
|
;
Liebner et al., 2004). The
R26R (Soriano, 1999)
allele was introduced into the cross for monitoring recombination efficiency
and specificity, as we observed unexpected ectopic recombination induced by
Tie2-Cre starting from E10.5. Approximately 30% of embryos at E10.5
displayed ectopic recombination throughout the whole embryo, as determined
from expression of the R26B reporter, whereas the remaining 70% of
embryos displayed the expected endothelium-specific recombination. No live
Tie2cre;Tgfbr2loxp/loxp;R26R embryos with ectopic
recombination were recovered beyond E11.5; the embryonic lethality is
presumably due to broad inactivation of Tgfbr2 throughout these
embryos. To overcome the ectopic recombination problem, embryos in this study
were stained with X-gal when possible, and only those with proper endothelium
specific recombination at the R26 locus were included for further
analysis.
Approximately 65% of the conditional knockout embryos (51/92; excluding
embryos with ectopic recombination) isolated from E9.0 to E11.5 were arrested
at the 20-25 somite stage (Fig.
2A-C). Their yolk sacs showed severe anemia and a deficiency in
vasculogenesis that was indistinguishable from that of
Tgfbr2-/- embryos, whose growth was arrested at the same
stage (Oshima et al., 1996).
No mutant embryo with apparent retardation was recovered beyond E11.5, and
demise was most likely to be secondary to yolk sac vascular insufficiency, as
previously described (Oshima et al.,
1996). However, the remaining
Tie2cre;Tgfbr2loxp/loxp;R26R embryos did not display
overall growth retardation or obvious abnormalities in their yolk sac
vasculatures, and were able to survive to the E12.5 stage
(Fig. 2D,E,
Fig. 3A). As the mice were from
a mixed genetic background, the mutant embryos with normal growth probably
have inherent beneficial genetic factors that influence Tgfß signaling in
vivo, similar to those previously described
(Tang et al., 2003).
Alternatively, there may have been a delay in Tgfbr2 excision in yolk
sac endothelia of this group of embryos. The Tie2cre-mediated
endothelial recombination at the R26R locus in these embryos was
indistinguishable from the control embryos, as determined from both
whole-mount and section studies (Fig.
3A,C-F), suggesting that the grossly normal growth of these mutant
embryos is unlikely to be caused by insufficient recombination in their
endothelium. No live mutant embryos between E13.5 and E15.5 (0/81) were
recovered. Dead mutant embryos were often found at E13.5 with hemorrhage and
edema, presumably caused by cardiovascular insufficiency (data not shown).
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|
Deletion of Tgfbr2 in endocardial cells blocks EMT in in vitro cultures
Because our results were contrary to previous in vitro studies, we
performed collagen gel assays, and found that very few mesenchymal cells were
formed in mutant AV explants (Fig.
4). In contrast to the well-formed mesenchymal cells in the
control explant (Fig. 4A), in
the mutants, endothelial cells were associated with neighbor cells, and were
expanded on the collagen gel but did not undergo activation or transformation
(Fig. 4B). This result was
confirmed by a quantitative assay (Fig.
4C), and by immunofluorescent studies
(Fig. 4D-G) using antibodies
against Pecam (an endothelium-specific marker) and -smooth-muscle actin
(a mesenchymal marker). Significantly, this phenotype is identical to the
result obtained in wild-type AV explant cultures treated with anti-Tgfß2
antiserum (Camenisch et al.,
2002), confirming that Tgfß signaling is required for EMT in
AV explant assays.
|
)]. This result is consistent with a previous study, which
showed that some Tgfb2-/- embryos displayed defects during
the second phase of looping (Bartram et
al., 2001). Section studies showed that the inferior cushion of the mutant hearts had a dramatically reduced size and failed to fuse with the mesenchymal cap of the atrial septum primum (ASP), whereas the size of the superior cushion appeared normal (Fig. 5C-H). The size reduction in the inferior cushion but not in the superior cushion was further confirmed with quantification analysis (Fig. 5I). Significantly, in contrast to the control hearts in which the left and right atria were connected separately with the left and right ventricles (Fig. 5C,F), both atria of the mutant hearts were open only to the LV (Fig. 5D,G,H) in all sections that were examined, resulting in a DILV defect. All mutant embryonic hearts (Fig. 5L) showed a ventricular septal defect (VSD), which is a hemodynamic necessity for DILV. The DILV defect was not due to underdevelopment of the RV (Fig. 5J-M), nor to abnormal yolk sac vasculogenesis (Fig. 2D,E). In addition, we observed defects in septation of the OFT in all mutant hearts. Detailed characterization of OFT and general endothelial defects is currently underway.
To test whether the uneven development of AV cushions (inferior cushion versus superior cushion) in mutant embryonic hearts is due to differential expression of Tgfbr2, we performed radioactive in situ hybridization analysis on sections of wild-type embryos from E9.5 to E11.5. As shown in Fig. 5N-Q, no obvious asymmetric expression of Tgfbr2 in AV cushions was observed, although we cannot rule out a potential subtle difference beyond our detection.
Inactivation of Tgfbr2 in endocardial cells preferentially reduces cell proliferation in the inferior AV cushion
We did not observe any enhanced apoptosis in the mutant embryonic hearts at
E11.5 and E12.5 (data not shown). To test whether proliferation of mesenchymal
cells was affected in AV cushions, we stained embryo sections with an
anti-Ki67 antibody (Fig. 6A-I).
Our result shows that the growth rate of inferior cushion mesenchyme of mutant
hearts was significantly reduced at E11.5, whereas that of the superior
cushion was normal. Thus, Tgfß signaling is required for the proper
inferior cushion mesenchyme proliferation.
To gain insight into the mechanism of Tgfß signaling in regulating the
AV mesenchyme cell cycle, we performed immunofluorescent studies with a cyclin
D1-specific antibody. Cyclin D1 promotes cell-cycle progression by regulating
Rb phosphorylation in nuclei (Sherr and
Roberts, 1999), and it was previously reported that elimination of
Tgfß signaling in palatal mesenchyme reduced cyclin D1 expression and
inhibited cell proliferation (Ito et al.,
2003). Using quantitative immunofluorescence analysis, we observed
a significant reduction in cyclin D1 expression in the inferior cushion but
not in the superior cushion of mutant hearts
(Fig. 6J-Q). This is consistent
with the observation that cell proliferation is only significantly reduced in
the inferior AV cushion. In addition to the overall cyclin D1 expression
reduction, we found that the percentage of mesenchyme with nuclear-localized
cyclin D1 is also significantly reduced in the inferior cushion of mutant
embryonic hearts (Fig. 6J-Q).
Cytoplasmic cyclin D1 is nonfunctional and targeted for proteolysis
(Alt et al., 2000;
Sherr and Roberts, 1999), and
cyclin D1 cytoplasmic sequestration serves as an important regulatory
mechanism for controlling cell proliferation, survival and fate determination
(see Sumrejkanchanakij et al.,
2003; Tamamori-Adachi et al.,
2003).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Dickson et al., 1995;
Kaartinen et al., 1995;
Sanford et al., 1997;
Stenvers et al., 2003). In
this study, we specifically inactivated Tgfbr2, which encodes the
only known type II receptor for Tgfß1-3 ligands, in the endothelium. Our
studies using the collagen gel system essentially confirm previous in vitro
studies in that no EMT occurs in AV explants of
Tie2cre;Tgfbr2loxp/loxp embryos
(Fig. 4). However, the current
study fails to provide evidence supporting an essential role of
Tgfbr2 during EMT in living embryos. We showed that transcription of
Tgfbr2 in mutant embryos is reduced to less than 10% of the wild-type
level and, because of to technical difficulties, we failed to detect Tgfbr2 in
AV cushions using the currently available antibody. Therefore, we cannot
exclude that residual Tgfbr2 can sufficiently activate EMT; however, the
failure in EMT observed from the collagen gel assay using mutant explants
provided functional evidence showing that the expression of Tgfbr2
was efficiently inactivated.
|
;
Sugi et al., 2004). It has
been reported that Bmp2 can induce EMT in in vitro culture assays
(Sugi et al., 2004) and,
furthermore, that myocardium-specific inactivation of Bmp2 blocks EMT
in mouse embryos (Ma et al.,
2005). We cannot exclude the possibility that Tgfß ligands
may act through other type II receptors (such as ActRIIB; Acvr2b - Mouse
Genome Informatics) when Tgfbr2 is absent in the AV cushions of live embryos,
although Tgfbr2 still remains the only known type II receptor for Tgfß
signaling that has been substantiated by clear experimental evidence. The
possibility that Tgfß1-3 ligands act through other type II receptors is
certainly an intriguing avenue of future investigation, but clearly beyond our
current knowledge. An alternative explanation to reconcile the discrepancy
between in vivo and in vitro studies is that although it is not absolutely
required for EMT, Tgfbr2 promotes survival and proliferation of
mesenchymal cells, and hence very few mesenchymal cells were observed in the
collagen gel system. Although we cannot fully exclude this explanation, we do
not favor it for the following reason: given the dramatic difference in the
number of mesenchymal cells observed in the collagen gel between control and
mutant explants (>20-fold; Fig.
4), the Tgfbr2-mediated Tgfß signaling would have a
very potent effect on mesenchymal cell proliferation and/or survival. In our
study, we did not observe increased apoptosis of AV mesenchymal cells in
mutant embryos. The reduction of cell proliferation in the AV cushions of
mutant embryos is relatively moderate (50% Ki67-positive nuclei in the
inferior cushions of controls versus 30% in mutants;
Fig. 6), and is limited to
within the inferior cushion, whereas both inferior and superior cushions were
included in a single explant in the collagen gel assay. Thus, the reduction in
cell proliferation is unlikely to account for the dramatic decrease of
mesenchymal cells in mutant explant cultures. Taken together, our results
suggest that complementary mechanisms exist for the loss of Tgfbr2 in
living embryos to support EMT. The collagen gel culture system may be
particularly useful in studying Tgfß signaling during EMT, as
compensatory pathways are not present to confound experimental results.
|
). We speculate that some morphogenic signaling molecules
expressed in the OFT, such as Bmp4, Bmp6, Bmp7 and Fgf8
(Delot, 2003;
Hu et al., 2004;
Jiao et al., 2003;
Kim et al., 2001b), can
diffuse into the superior cushion to compensate for the loss of Tgfß
signaling, but are inaccessible to the inferior cushion.
This study describes a unique mouse genetic model with the DILV defect.
This form of CHD accounts for the most common type of single ventricle
physiology (Vanpraagh et al.,
1965). The defect results from the failure to establish a direct
communication between the right atrium and the RV, a crucial step for
transforming the single-channeled cardiac primordia into a mature
double-channeled organ. Formation of the right-chamber connection is achieved
through the rightward expansion of the AVC in human embryos from 6.5 to 7
weeks (Kim et al., 2001a) -
corresponding to E11.5-E12.5 in mice. The involvement of Tgfß
signaling in alignment between atria and ventricles was first demonstrated in
a study of Tgfb2 knockout mice; 25% of the
Tgfb2-/- embryos demonstrated an overriding of the
tricuspid valve (Bartram et al.,
2001; Sanford et al.,
1997), which may be considered as a mild phenotype in the spectrum
of DILV. However, it is unclear from the above studies what is the primary
defective cell population responsible for the malalignment defect. Although
the development of AVC myocardium was thought to be the primary driving force
for this rightward expansion (Kim et al.,
2001a), the fact that endocardial but not myocardial depletion of
Tgfbr2 (Fig. 1)
results in DILV demonstrates for the first time that misalignment between
atria and ventricles can be primarily caused by impaired Tgfß signaling
in the valvular mesenchyme derived from endocardium. We propose that the
unbalanced growth between the inferior and superior AV cushions in mutant
embryos distorts the AVC, leading to aberrant cardiac looping during the
second phase, and at least partially contributes to DILV. Alternatively,
Tgfbr2 has distinct roles in promoting mesenchyme proliferation in the
inferior AV cushion and in patterning embryonic hearts for proper looping.
In summary, in vivo tissue-specific inactivation of Tgfbr2 identifies a novel role for Tgfbr2 in stimulating mesenchymal cell proliferation in the inferior cushion through regulating the expression and subcellular localization of cyclin D1, which may be important for understanding the mechanisms related to the development of DILV and other pathologies involving unbalanced ventricular inflow. Our study does not support an essential role for Tgfbr2-mediated Tgfß signaling either in myocardial development or during initial EMT.
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ACKNOWLEDGMENTS |
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