|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online January 26, 2007
doi: 10.1242/10.1242/dev.02777
1 Division of Mammalian Development, National Institute of Genetics, 1111 Yata,
Mishima Shizuoka 411-8540, Japan.
2 Department of Genetics, The Graduate University for Advanced studies, 1111
Yata, Mishima Shizuoka 411-8540, Japan.
3 Department of Pediatric Cardiology, The Heart Institute of Japan, Tokyo
Women's Medical University, 8-1 Kawada-cho, Shinjyuku-ku, Tokyo 162-8666,
Japan.
4 National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki Aichi
444-8585 Japan.
* Authors for correspondence (e-mail: hkokubo{at}lab.nig.ac.jp; ysaga{at}lab.nig.ac.jp)
Accepted 6 December 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Hesr1 (Hey1), Hesr2 (Hey2), Heart, Notch signaling
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Delamination and migration of endothelial cells to these
cushions occurs by an epithelial-to-mesenchymal transformation (EMT). As the
cushions expand, anlages of the septal and valvular structures are formed to
demarcate the developing chambers.
Several transcription factors have now been implicated in both atrial and
ventricular chamber formation in the mouse. Knockout (KO) studies in mice for
the Nkx2-5, Tbx5, Mef2c, Hand2, and Tbx20 genes indicate
that these factors may be involved in chamber specification. The continuous
expression of these factors throughout the cardiac tube, however, has not
resulted in the identification of the genes responsible for specification. In
addition, although the expression of Irx4 is restricted to the
ventricular chamber (Yamagishi et al.,
2001), Irx4-deficient mice are viable at adulthood,
although they develop impaired contractile function
(Bruneau et al., 2001).
Hand1 and Cited1 are expressed in a ventricle-specific
manner at an early stage of development
(Carotta et al., 1998;
Thomas et al., 1998), and
Hand1 has been suggested to be required for the proper formation of
the outer curvature of the ventricle and interventricular septum
(Togi et al., 2004), but not
the atrioventricular (AV) boundary. Hence, little is known about the
mechanisms that control the differentiation of the cardiogenic precursors and
their acquisition of atrial and ventricular chamber-specific properties.
The myocardium of the AV canal (AV myocardium) is important for the
development of the AV cushion and AV node. Bone morphogenetic protein 2
(Bmp2) is expressed in the AV myocardium and conditional KO studies
of the Bmp2 gene in the mouse cardiac lineage suggest that it is
involved in cardiac cushion development, including the induction of EMT, the
accumulation of cardiac jelly and the establishment of AV canal specificity
(Zhang and Bradley, 1996;
Ma et al., 2005). Tbx2, a
T-box transcription factor, is also expressed in the AV myocardium. Tbx2 is
induced by chicken Bmp2 beads implanted in the AV myocardium in chick, and
shows reduced expression in Bmp2-null mice. These observations
indicate that Bmp2 establishes the AV cushions via the induction of
Tbx2 (Yamada et al.,
2000; Ma et al.,
2005). Tbx2-null mice also show morphological defects in
the AV canal and allow the expression of chamber differentiation markers in
the AV myocardium, indicating that Tbx2 is required to repress chamber
differentiation in the AV canal region
(Harrelson et al., 2004).
However, the mechanisms underlying the formation of the boundaries between the
chambers and the AV canal remain unclear.
The Hesr genes (also known as Hey, Hrt, Chf, Herp or
Gridlock) were identified through their similarities to the hairy and
enhancer of split (Hes) family of genes
(Kokubo et al., 2005a).
Several previous studies have also now shown that the Hesr genes are
putative direct targets of Notch signaling
(Iso et al., 2003). Because
the myocardial expression of Hesr1 (Hey1) or Hesr2
(Hey2) is restricted to the atrium and ventricle, respectively, it
has been speculated that these genes might regulate atrial or ventricular
specification. However, Hesr1-null mice show no obvious phenotype in
their cardiac development and Hesr2-null mice display no anomalies
during chamber specification, although several defects do occur in these
animals, including dysplastic AV valves, a perimembranous ventricular septal
defect, and a secundum atrial septal defect
(Donovan et al., 2002;
Gessler et al., 2002;
Sakata et al., 2002;
Kokubo et al., 2004). Even in
Hesr1-Hesr2 double-null mice, the atria and ventricles seem to be
properly formed, although they do show some defects during cardiac
development, including few EMT cells in the AV cushions and abnormal apoptosis
in the trabecular layer of the ventricle.
To further clarify the function of Hesr1 and Hesr2 during cardiac development, we have established a system whereby Hesr1 and Hesr2 are constitutively expressed in the cardiac lineage in mouse. This enables us to analyze the functions of Hesr1 and Hesr2 during chamber specification. Marker analyses reveal a significant reduction in and loss of the AV canal in the Hesr1- and Hesr2-misexpressing (ME) hearts, respectively. Furthermore, by analysis of the respective KO animals, as well as of the corresponding ME hearts, we find that Hesr1 and Hesr2 can directly repress the expression of Tbx2. These observations indicate that Hesr1 and Hesr2 play essential roles during AV boundary formation through the repression of Tbx2.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
The lacZ expression vectors, Tbx2-D3-nlacZ and
Tbx2-Xho-nlacZ, were constructed by insertion of the 6083 bp
(HindIII-NcoI) or 2712 bp (XhoI-NcoI)
upstream region of the Tbx2 gene, derived from the BAC clone
RP23-48A17, into a lacZ cassette
(Kokubo et al., 2005b). These
constructs were injected into fertilized eggs to generate permanent transgenic
lines by standard methods. Each transgenic line was then crossed with a
Mesp1-Cre mouse line
(Saga et al., 1999) to obtain
embryos expressing the inserted cDNAs in the cardiac lineage. We refer to such
gene-misexpressing mice as ME mice.
Histological analysis
Histological observations, Hematoxylin and Eosin staining, and transmission
electron microscopic analyses were carried out as described previously
(Miyagawa-Tomita et al.,
1996). The InsituPro system (M&S Instruments) was used for
whole-mount in situ hybridizations according to the manufacturer's
instructions. Section in situ hybridizations were performed using 20 µm
frozen sections. Immunohistochemistry was performed using anti-myosin
(Skeletal, Slow; Sigma) and anti-
-smooth muscle actin (IA4; Sigma)
antibodies with 6 µm paraffin sections using standard protocols.
Quantification of relative expression domains
The ratio of the Bmp2-expressing to non-expressing area in the
heart was determined from a lateral side view using the WinRoof program
(Mitani Corp, Japan) (n=4).
RT-PCR analysis
Total RNA was extracted from mouse hearts (n=10) using a
mini-extraction kit (Qiagen). Real time quantitative PCR (RT-PCR) was then
performed using the ExTaq-RPCR kit (Takara) with the MiniOpticon RT-PCR system
(Bio-Rad). The primers and PCR conditions for the genes under study have been
described previously (Watanabe et al.,
2006).
Luciferase assay
For luciferase reporter analysis of the 6 kb
(HindIII-NcoI) and 2.7 kb (XhoI-NcoI)
Tbx2 upstream enhancer regions, reporter constructs (200 ng) were
individually transfected with or without expression vectors for the
constitutively active or kinase-dead forms of Alk3 (50 ng), 3xFlag-Smad5
(20 ng), 6xMyc-tagged Hesr1 (10-50 ng) or 6xMyc-tagged Hesr2
(10-50 ng). Transfections of NIH3T3 cells (0.25x105 cells per
well in 24-well plates) were then performed using Lipofectamine Plus
(Invitrogen), according to the manufacturer's instructions. The vector
containing the Renilla luciferase gene under the control of the thymidine
kinase promoter (10 ng) was used as an internal standard to normalize for
transfection efficiency. After 36 hours of further culturing, cell lysates
were prepared and the luciferase activities measured using the Dual Luciferase
Assay Kit (Promega).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Expansion of the AV canal in Hesr1 and Hesr2 individual- and double-KO hearts
Given that the members of the Hesr family function as transcriptional
repressors, we hypothesized that both Hesr1 and Hesr2 would suppress genes
that are essential for AV canal formation. Because Bmp2 is
specifically expressed in the AV myocardium in wild-type embryos and its
conditional KO results in the reduced formation of the AV canal
(Ma et al., 2005), we
speculated that Hesr1 and Hesr2 might function in the formation of the AV
boundaries by repressing Bmp2. If this proved to be the case, we
predicted that in the absence of Hesr1 or Hesr2, Bmp2 expression
would be expanded to the atrium or ventricle. In our Hesr1-null
embryos, the expression of Bmp2 was specifically detected in the AV
canal, and was expanded compared with the wild-type heart
(Fig. 1, compare H with G). The
Bmp2 expression area representing the AV canal was also increased by
approximately 11%, judged from a lateral side view
(Fig. 1K). This finding was
supported by the expression pattern of Anf (Nppa - Mouse
Genome Informatics), which is a chamber-specific marker
(Fig. 1, compare M with L).
Similarly, an expanded AV canal showing Bmp2 and Anf
expression was observed in both Hesr2-null and Hesr1-Hesr2
double-null embryos (Fig. 1, compare
I,J,N,O with G,L). However, this expansion showed no tendency
towards the atrium or ventricle in either the Hesr1- or
Hesr2-null heart.
We also examined whether the expression of Tbx2, which is a known downstream target of Bmp2, is affected in these KO mutants. Tbx2 was specifically detected in the AV myocardium in wild-type embryos, but was found to be slightly expanded to the atrium of the Hesr1 KO hearts (arrowhead in Fig. 1Q). Tbx2 is weakly expressed but was found to be extended to the ventricle in the Hesr2 KO hearts (arrowhead in Fig. 1R). In addition, ambiguous boarders for the AV canal were observed in the double-KO heart (arrowheads in Fig. 1S). These observations suggest that Hesr1 and Hesr2 are involved in AV canal specification, although no definitive conclusions could be drawn in this regard from these loss-of-function studies.
Generation of mice that express Hesr1 and Hesr2 in the entire cardiac lineage
As an alternative method of revealing the possible functions of Hesr1 and
Hesr2 during regional specification and/or AV boundary formation, we attempted
the forced expression of these genes throughout the entire mouse heart. We
created the CAG-lox-CAT-lox-Hesr1 and -Hesr2
transgenic lines, which begin expressing these genes under the control of the
CAG promoter upon excision of the CAT gene by Cre recombinase. For
this purpose, we crossed these transgenic lines with an Mesp1-Cre
knock-in mouse line, which shows transient expression of Cre in
cardiovascular precursor cells at the cylinder stage (E6.5). Using this
transgenic strategy, we successfully obtained embryos showing the expression
of Hesr1 (Fig. 2A) or
Hesr2 (Fig. 2B)
throughout the cardiac lineage. Section in situ analysis confirmed the
expression of these genes in the myocardium of the entire heart, although this
was not observed clearly in the endocardial cells (see Fig. S1 in the
supplementary material). We used RT-PCR to measure the expression levels of
the Hesr1 and Hesr2 genes using RNA prepared from pooled
hearts (n=10) generated from two independent transgenic lines for
each gene. We found that the expression levels were elevated 2.5- to 5.3-fold
relative to wild type (Fig.
2C). As the gross morphological phenotypes were similar in the two
lines for each gene, we utilized Hesr1-ME-Line#2 and
Hesr2-ME-Line#2 for further analysis.
|
Until stage E9.5, both Hesr1- and Hesr2-ME embryos appear to develop normally. However, the Hesr1-ME embryos die by E11.5, showing heart malformations. Hesr2-ME embryos die at around E10, harboring not only cardiac defects but also vascular defects (data not shown). Histological examination of wild-type embryos at E9.5 shows that the atrium, the AV canal, and the ventricle of the heart are well developed, and that the endocardium of the inferior- and superior-AV cushions are attached to each other and begin undergoing EMT (Fig. 2F). In the Hesr1-ME embryo, however, the atrium and the ventricle appear to be normal but the width of the AV canal, recognized by the characteristic constriction between the atrium and the ventricle, is found to be small (bracket in Fig. 2G). Despite the small width of the AV canal in the transgenic embryo, the endocardial cushion tissue is still formed, and mesenchymal cells that have undergone EMT are detectable. Strikingly, in the Hesr2- ME embryos, AV canal constriction is rarely observed and the atrium and ventricle seemed to be directly connected (arrowhead in Fig. 2H). In this region, however, neither the accumulation of cardiac jelly nor EMT were observed, indicating that the myocardium of the AV canal is not formed.
It is also noteworthy that the trabeculation of the ventricle is not well
developed in the Hesr2-ME embryo
(Fig. 2H). We therefore
performed TEM analysis of myocardium in the Hesr1- and
Hesr2- ME embryos at E9.5. At this stage, in the wild-type embryo the
myocardial cells develop thick myofibrils with clear sarcomere structures, in
which the Z bands were clearly visible
(Fig. 2I). In both
Hesr1- and Hesr2-ME myocardial cells, however, only
disorganized thin myofibrils develop (Fig.
2J,K), in which the Z bands were often barely detectable,
indicating immature myocardial differentiation in both transgenic mouse types.
We next analyzed the expression of early markers for myocardial
differentiation - myosin light chain (Mlc2v; Myl2 - Mouse
Genome Informatics), Mlc2a (Myl7 - Mouse Genome
Informatics), -smooth muscle actin, and myosin heavy chain - and
Bmp10 for ventricular trabeculation. None of these markers were found
to be altered at the E9.5 stage in the Hesr1- or Hesr2-ME
hearts (data not shown), indicating that early myocardial differentiation and
trabeculation occur normally in these ME embryos.
|
A similar phenomenon was also observed for the chamber-specific genes, connexin 40 (Cx40; Gja5 - Mouse Genome Informatics) (Fig. 3G-I), Anf (see Fig. S2D-F in the supplementary material) and Chisel (Smpx - Mouse Genome Informatics) (see Fig. S2G-I in the supplementary material), which are normally expressed in both the atrium and ventricle at this stage in the wild-type embryo (Fig. 3G, and see Fig. S2D,G in the supplementary material). In the Hesr1-ME heart, a normal expression pattern for these genes was observed in both the atrium and ventricle, but the expression borders along the AV canal become ambiguous (arrowheads in Fig. 3H, and see Fig. S2E,H in the supplementary material). Strikingly, in the Hesr2-ME heart, the expression of each of these genes was found to be continuous from the atrium to the ventricle (arrowheads in Fig. 3I, and see Fig. S2F,I in the supplementary material). These observations suggest that the boundaries between the AV canal and the chambers are not clearly established in the Hesr1-ME heart, and that the AV canal does not form in the Hesr2-ME heart.
Bmp2 and Tbx2 are downregulated in both the Hesr1- and Hesr2-misexpressing hearts
To next investigate the formation of the AV canal in greater detail in the
Hesr1- and Hesr2-ME hearts, we examined the expression of
the AV myocardial factors Bmp2 and Tbx2
(Fig. 4A-F). In
Hesr1-ME embryos, Bmp2 is weakly expressed
(Fig. 4B) and Tbx2 is
strongly downregulated (Fig.
4E), suggesting that the suppression of Tbx2 is one of
main causes of the AV cushion phenotype in these embryos. Interestingly, the
Hesr2-ME embryos showed loss of both Bmp2 and Tbx2
expression in the AV canal (Fig.
4C,F), indicating that the repression of not only Tbx2
but also Bmp2 may lead to loss of the AV canal in these embryos.
Hesr1 and Hesr2 may directly repress Tbx2 expression
Because our misexpression studies strongly suggest that Tbx2
expression is suppressed by both Hesr1 and Hesr2, we performed experiments to
determine the location of a Tbx2 enhancer likely to be involved in
this mechanism. We created two kinds of LacZreporter transgenic line,
containing either 2.7 kb (Tbx2-Xho-nlacZ) or 6 kb
(Tbx2-D3-nlacZ) of the region upstream of the Tbx2
translational start site (Fig.
4O). Two independent Tbx2-Xho-nlacZ lines were
found to show no ß-gal expression in the AV canal
(Fig. 4G,H). By contrast, two
independent Tbx2-D3-nlacZ lines showed positive expression in not
only the AV canal, but also in the outflow tract and eyes
(Fig. 4I,J). Moreover, this
latter expression pattern is similar to that of the endogenous Tbx2
gene. We next examined the expression of a Tbx2 transgene in both the
Hesr1- and Hesr2-ME background by generating triple
transgenic mice
(Tbx2-D3-nlacZ/Mesp1-Cre/CAG-CAT-Hesr1 or
-Hesr2). As expected, the ß-galactosidase expression pattern was
greatly reduced in the AV canal, but not in the eyes, of both Hesr1-
and Hesr2-ME embryos (Fig.
4K-N), indicating that the cis-regulatory elements that are
required for the repression of Tbx2 by Hesr1 and/or Hesr2 exist in
the HindIII-XhoI upstream region of this gene. However, we
could not exclude the possibility that the downregulation of Tbx2 is
a consequence of the suppression of Bmp2, particularly in the
Hesr2-ME embryos. To address this possibility, we established a
luciferase reporter assay system using NIH3T3 cells and a reporter construct
containing the 6 kb upstream region of Tbx2 (Tbx2-D3-Luc).
This reporter showed little response to a constitutively active form of Alk3
(Bmpr1a - Mouse Genome Informatics), a receptor of Bmp2, but exhibited
upregulation upon the addition of Smad5, which is suppressed by both Hesr1 and
Hesr2 (Fig. 4P). These data
suggest that Tbx2 transcription, which is upregulated by Bmp
signaling, is suppressed by both Hesr1 and Hesr2. However, it has been
suggested that several putative Smad-binding sites, but not Hesrbinding sites,
are localized within a 
400 bp stretch of the
HindIII-XhoI upstream region, and this possibly serves as
the enhancer of Tbx2 that drives AV canal restricted expression (M.
Shirai, personal communication). This observation suggests that the
Hesr genes repress Tbx2 expression through mechanisms that
are independent of DNA binding.
|
). We
thus examined whether Tbx2 would downregulate the expression of Hesr1
and/or Hesr2 in the AV myocardium, and thereby form a
negative-feedback system that would establish the precise boundaries for the
atrium and ventricle in mice. To address this possibility, we established a
Tbx2-misexpression transgenic mouse line. However, our observations
revealed a normal expression pattern for Hesr1 and Hesr2 in
the Tbx2-ME heart (Fig.
5A-C), suggesting that Hesr1 and Hesr2 are not
repressed by Tbx2.
The myocardial expression of Hesr1 and Hesr2 is not regulated by Notch2
Hesr1 and Hesr2 are thought to be direct downstream
targets of Notch signaling in culture systems
(Iso et al., 2003). However,
the expression of Notch1 and Notch4 is restricted to the endocardium and does
not occur in the myocardium, suggesting that the myocardial expression of
Hesr1 and Hesr2 is unlikely to be regulated by either of
these Notch factors. However, as Notch2 is expressed in the myocardium of the
heart we examined whether it might be an upstream regulator of Hesr1
and/or Hesr2. A normal expression pattern for Hesr1 and
Hesr2 was found specifically in the atrium and ventricle of the
Notch2-KO mouse (Fig.
5D,E). We also generated transgenic lines for the misexpression of
Notch2 activated form, which express only the intracellular domain
(IDC) of Notch2 (Fig.
5F,I), using a similar strategy to that mentioned above. However,
both the atrial- and ventricular-specific expression patterns of
Hesr1 or Hesr2 were unaffected in the Notch2-ICD-ME
hearts (Fig. 5G,H). These data
suggest that the expression of Hesr1 and Hesr2 is regulated
in the myocardium through a Notch2-independent pathway.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
). In
Hesr1- or Hesr2-KO mice, and in the Hesr1-Hesr2
double-KO mice, an expanded AV canal was observed as an extended expression
region of Bmp2, although this expansion did not have any bias to
either the atrial or ventricular chamber. These data suggest that neither
Hesr1 nor Hesr2 is the direct repressive regulator of Bmp2. However,
Tbx2 expression was found to be expanded to the atrial side in the
Hesr1-null embryos and to the ventricular side in the
Hesr2-null embryos (Fig.
1Q,R). We also observed repressive effects of both Hesr1 and Hesr2
in a luciferase reporter assay using upstream Tbx2 enhancer
sequences. We thus speculate that the Bmp proteins secreted by the AV
myocardium induce Tbx2 expression in this structure and also in the
surrounding cells, if the Hesr genes are not expressed. Hesr1 or
Hesr2 must therefore suppress Tbx2 expression in the atrium or
ventricle to define the boundaries between the atrium and the AV canal (by
Hesr1), and ventricle and AV canal (by Hesr2), during normal development.
However, the repression of Tbx2 is not the only mechanism responsible
for the lack of an AV canal because the AV canal is present in the
Tbx2-null heart (Harrelson et
al., 2004).
Similarly, the heart-specific knockout of Bmp2 results in an
abnormal AV canal constriction morphology, but the AV canal itself is not
completely lost (Ma et al.,
2005). The reason for the complete lack of the AV canal in the
Hesr2-ME heart is currently unknown, yet unidentified upstream
regulator(s) for AV canal formation must be regulated by Hesr2. In this
regard, the function of Tbx20, which is expressed throughout the entire heart,
should be considered. Recently, Tbx20 was reported to be essential for chamber
differentiation, as the loss of this gene results in the downregulation of
chamber-specific markers, including Hesr1 and Hesr2, whereas
the expression of the AV canal marker Tbx2 was found to be expanded
(Cai et al., 2005;
Singh et al., 2005;
Stennard et al., 2005). Hence,
Tbx20 is one of the positive upstream regulators of Hesr1 and
Hesr2, although it is not known how Hesr1 and Hesr2
are restricted in the atrium or ventricle. There cannot be feedback regulation
of Tbx20 by Hesr1 and Hesr2 as we did not observe any notable changes
in its expression pattern in the Hesr1- and Hesr2-ME hearts
(see Fig. S2J-L in the supplementary material).
The mechanisms underlying the establishment of the regional specificity and
the lineage origin of the cells that eventually form the heart remain to be
elucidated. Recently, however, it has been shown that myocardial cells from a
second source make an important contribution to the cardiac chambers
(Buckingham et al., 2005). Gene
expression lineage tracing data show that Isl1 is expressed in a more
anterior/medial field relative to the cells expressing Mlc2a at E7.5, and also
that these cells are added mainly to the right ventricle and outflow tract.
Although the cells expressing Mlc2a have been suggested to contribute to the
left ventricle, the lineage origin of the atrium and AV canal remains
ambiguous. Several genes, which have been shown to mark the nascent chamber
myocardium, begin to be expressed only after the chambers are clearly visible
at E9.5. We therefore expect that future detailed analysis of the early
expression of Hesr1 and Hesr2, in comparison with the
Isl1 or Mlc2a genes, will provide precise fate-mapping
information that could help to delineate the heart chambers.
|
|
;
Doi et al., 2005;
Garg et al., 2005). Therefore,
if Hesr2 but not Hesr1 interacts with such an activator(s), the development of
different phenotypes would be expected in transgenic studies. Alternatively,
both Hesr1 and Hesr2 might interact with the same activator but with a
different affinity, which could also account for the different transgenic
phenotypes. Further experiments will be necessary to elucidate the functional
differences between Hesr1 and Hesr2.
The role of the Notch signaling pathway
The expression of Hesr1 and Hesr2 has been shown
previously to be regulated by Notch signaling
(Maier and Gessler, 2000;
Nakagawa et al., 2000;
Iso et al., 2003). Notch2 is
the only Notch receptor expressed in the myocardium, but in our current study
we found that the myocardial expression of Hesr1 and Hesr2
is unaffected in both the Notch2-KO and Notch2-activated
embryonic mouse heart. These observations suggest that Hesr1 and Hesr2 play
crucial roles in AV boundary formation through a Notch2-independent pathway.
In the chick, however, Hesr1, but not Hesr2, has been
reported to respond to Notch2 (Rutenberg
et al., 2006). As Hesr1 expression in the ventricle is
observed in the chick but not in mice, the responsiveness of this gene to
Notch2 might have diverged during evolution. Previously, we reported that the
forced expression of Notch1-ICD results in the upregulation of
Hesr1 but not Hesr2 expression
(Watanabe et al., 2006). In
addition, Hesr1 but not Hesr2 expression in the heart is
downregulated in RBPJ
(Rbpsuh - Mouse Genome Informatics)
-null mutant mice (Timmerman et al.,
2004), indicating at least that Hesr1 but not
Hesr2 is regulated by RBPJ-dependent Notch1 signaling.
However, Notch1, Notch2 and Notch4 are expressed in the endocardial cells of
the AV cushion tissue, and expression of Hesr1 and Hesr2
also overlaps with this region. Therefore, Hesr1 and Hesr2
might be regulated by Notch signaling in the endocardial cells of the AV
cushion tissue, and thus might function in the EMT process. Further analyses
using cell-type specific KO or misexpression studies will be necessary to gain
a greater understanding of these signaling networks.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/4/747/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bruneau, B. G., Bao, Z. Z., Fatkin, D., Xavier-Neto, J.,
Georgakopoulos, D., Maguire, C. T., Berul, C. I., Kass, D. A., Kuroski-de
Bold, M. L., Conner, D. A. et al. (2001). Cardiomyopathy in
Irx4-deficient mice is preceded by abnormal ventricular gene expression.
Mol. Cell. Biol. 21,1730
-1736.
Buckingham, M., Meilhac, S. and Zaffran, S. (2005). Building the mammalian heart from two sources of myocardial cells. Nat. Rev. Genet. 6, 826-835.[CrossRef][Medline]
Cai, C.-L., Zhou, W., Yang, L., Bu, L., Qyang, Y., Zhang, X.,
Li, X., Rosenfeld, M. G., Chen, J. and Evans, S. (2005).
T-box genes coordinate regional rates of proliferation and regional
specification during cardiogenesis. Development
132,2475
-2487.
Carotta, M. C., Martinelli, G., Sadaoka, Y., Nunziante, P., Traversa, E., Dunwoodie, S. L., Rodriguez, T. A. and Beddington, R. S. P. (1998). Msg1 and Mrg1, founding members of a gene family, show distinct patterns of gene expression during mouse embryogenesis. Mech. Dev. 72,27 -40.[CrossRef][Medline]
Chin, M. T., Maemura, K., Fukumoto, S., Jain, M. K., Layne, M.
D., Watanabe, M., Hsieh, C. M. and Lee, M. E. (2000).
Cardiovascular basic helix loop helix factor 1, a novel transcriptional
repressor expressed preferentially in the developing and adult cardiovascular
system. J. Biol. Chem.
275,6381
-6387.
Doi, H., Iso, T., Yamazaki, M., Akiyama, H., Kanai, H., Sato,
H., Kawai-Kowase, K., Tanaka, T., Maeno, T., Okamoto, E.-i. et al.
(2005). HERP1 inhibits myocardin-induced vascular smooth muscle
cell differentiation by interfering with SRF binding to CArG box.
Arterioscler. Thromb. Vasc. Biol.
25,2328
-2334.
Donovan, J., Kordylewska, A., Jan, Y. N. and Utset, M. F. (2002). Tetralogy of fallot and other congenital heart defects in hey2 mutant mice. Curr. Biol. 12,1605 -1607.[CrossRef][Medline]
Eisenberg, L. M. and Markwald, R. R. (1995).
Molecular regulation of atrioventricular valvuloseptal morphogenesis.
Circ. Res. 77,1
-6.
Garg, V., Muth, A. N., Ransom, J. F., Schluterman, M. K., Barnes, R., King, I. N., Grossfeld, P. D. and Srivastava, D. (2005). Mutations in NOTCH1 cause aortic valve disease. Nature 437,270 -274.[CrossRef][Medline]
Gessler, M., Knobeloch, K. P., Helisch, A., Amann, K., Schumacher, N., Rohde, E., Fischer, A. and Leimeister, C. (2002). Mouse gridlock. No aortic coarctation or deficiency, but fatal cardiac defects in Hey2 -/- mice. Curr. Biol. 12,1601 -1603.[CrossRef][Medline]
Harrelson, Z., Kelly, R. G., Goldin, S. N., Gibson-Brown, J. J.,
Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (2004).
Tbx2 is essential for patterning the atrioventricular canal and for
morphogenesis of the outflow tract during heart development.
Development 131,5041
-5052.
Iso, T., Kedes, L. and Hamamori, Y. (2003). HES and HERP families: multiple effectors of the Notch signaling pathway. J. Cell. Physiol. 194,237 -255.[CrossRef][Medline]
Kokubo, H., Miyagawa-Tomita, S., Tomimatsu, H., Nakashima, Y.,
Nakazawa, M., Saga, Y. and Johnson, R. L. (2004). Targeted
disruption of hesr2 results in atrioventricular valve anomalies that lead to
heart dysfunction. Circ. Res.
95,540
-547.
Kokubo, H., Miyagawa-Tomita, S. and Johnson, R. L. (2005a). Hesr, a mediator of the Notch signaling, functions in heart and vessel development. Trends Cardiovasc. Med. 15,190 -194.[CrossRef][Medline]
Kokubo, H., Miyagawa-Tomita, S., Nakazawa, M., Saga, Y. and Johnson, R. L. (2005b). Mouse hesr1 and hesr2 genes are redundantly required to mediate Notch signaling in the developing cardiovascular system. Dev. Biol. 278,301 -309.[CrossRef][Medline]
Ma, L., Lu, M. F., Schwartz, R. J. and Martin, J. F.
(2005). Bmp2 is essential for cardiac cushion
epithelial-mesenchymal transition and myocardial patterning.
Development 132,5601
-5611.
Maier, M. M. and Gessler, M. (2000). Comparative analysis of the human and mouse Hey1 promoter: Hey genes are new Notch target genes. Biochem. Biophys. Res. Commun. 275,652 -660.[CrossRef][Medline]
Miyagawa-Tomita, S., Morishima, M., Nakazawa, M., Mizutani, M. and Kikuchi, K. (1996). Pathological study of Japanese quail embryo with acid alpha-glucosidase deficiency during early development. Acta Neuropathol. 92,249 -254.[CrossRef][Medline]
Nakagawa, O., McFadden, D. G., Nakagawa, M., Yanagisawa, H., Hu,
T., Srivastava, D. and Olson, E. N. (2000). Members of the
HRT family of basic helix-loop-helix proteins act as transcriptional
repressors downstream of Notch signaling. Proc. Natl. Acad. Sci.
USA 97,13655
-13660.
Rutenberg, J. B., Fischer, A., Jia, H., Gessler, M., Zhong, T.
P. and Mercola, M. (2006). Developmental patterning of the
cardiac atrioventricular canal by Notch and Hairy-related transcription
factors. Development
133,4381
-4390.
Saga, Y., Miyagawa-Tomita, S., Takagi, A., Kitajima, S., Miyazaki, J. and Inoue, T. (1999). MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development 126,3437 -3447.[Abstract]
Sakai, K. and Miyazaki, J. (1997). A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. Biochem. Biophys. Res. Commun. 237,318 -324.[CrossRef][Medline]
Sakata, Y., Kamei, C. N., Nakagami, H., Bronson, R., Liao, J. K.
and Chin, M. T. (2002). Ventricular septal defect and
cardiomyopathy in mice lacking the transcription factor CHF1/Hey2.
Proc. Natl. Acad. Sci. USA
99,16197
-16202.
Singh, M. K., Christoffels, V. M., Dias, J. M., Trowe, M.-O.,
Petry, M., Schuster-Gossler, K., Bürger, A, Ericson, J. and Kispert,
A. (2005). Tbx20 is essential for cardiac chamber
differentiation and repression of Tbx2. Development
132,2697
-2707.
Stennard, F. A., Costa, M. W., Lai, D., Biben, C., Furtado, M.
B., Solloway, M. J., McCulley, D. J., Leimena, C., Preis, J. I., Dunwoodie, S.
L. et al. (2005). Murine T-box transcription factor Tbx20
acts as a repressor during heart development, and is essential for adult heart
integrity, function and adaptation. Development
132,2451
-2462.
Thomas, T., Yamagishi, H., Overbeek, P. A., Olson, E. N. and Srivastava, D. (1998). The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness. Dev. Biol. 196,228 -236.[CrossRef][Medline]
Timmerman, L. A., Grego-Bessa, J., Raya, A., Bertran, E.,
Perez-Pomares, J. M., Diez, J., Aranda, S., Palomo, S., McCormick, F.,
Izpisua-Belmonte, J. C. et al. (2004). Notch promotes
epithelial-mesenchymal transition during cardiac development and oncogenic
transformation. Genes Dev.
18, 99-115.
Togi, K., Kawamoto, T., Yamauchi, R., Yoshida, Y., Kita, T. and
Tanaka, M. (2004). Role of Hand1/eHAND in the dorso-ventral
patterning and interventricular septum formation in the embryonic heart.
Mol. Cell. Biol. 24,4627
-4635.
Watanabe, Y., Kokubo, H., Miyagawa-Tomita, S., Endo, M.,
Igarashi, K., Aisaki, K.-i., Kanno, J. and Saga, Y. (2006).
Activation of Notch1 signaling in cardiogenic mesoderm induces abnormal heart
morphogenesis in mouse. Development
133,1625
-1634.
Yamada, M., Revelli, J.-P., Eichele, G., Barron, M. and Schwartz, R. J. (2000). Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2. Dev. Biol. 228,95 -105.[CrossRef][Medline]
Yamagishi, H., Yamagishi, C., Nakagawa, O., Harvey, R. P., Olson, R. N. and Srivastava, D. (2001). The combinatorial activities of Nkx2.5 and dHAND are essential for cardiac ventricle formation. Dev. Biol. 239,190 -203.[CrossRef][Medline]
Zhang, H. and Bradley, A. (1996). Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 122,2977 -2986.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  D. A. Venkatesh, K.-S. Park, A. Harrington, L. Miceli-Libby, J. K. Yoon, and L. Liaw Cardiovascular and Hematopoietic Defects Associated With Notch1 Activation in Embryonic Tie2-Expressing Populations Circ. Res., August 15, 2008; 103(4): 423 - 431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Niessen and A. Karsan Notch Signaling in Cardiac Development Circ. Res., May 23, 2008; 102(10): 1169 - 1181. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. C. Chi, R. M. Shaw, S. De Val, G. Kang, L. Y. Jan, B. L. Black, and D. Y.R. Stainier Foxn4 directly regulates tbx2b expression and atrioventricular canal formation Genes & Dev., March 15, 2008; 22(6): 734 - 739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Klaus, Y. Saga, M. M. Taketo, E. Tzahor, and W. Birchmeier Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis PNAS, November 20, 2007; 104(47): 18531 - 18536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Jia, I. N. King, S. S. Chopra, H. Wan, T. T. Ni, C. Jiang, X. Guan, S. Wells, D. Srivastava, and T. P. Zhong Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5 PNAS, August 28, 2007; 104(35): 14008 - 14013. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Fischer and M. Gessler Delta Notch and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors Nucleic Acids Res., July 14, 2007; 35(14): 4583 - 4596. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Xin, E. M. Small, E. van Rooij, X. Qi, J. A. Richardson, D. Srivastava, O. Nakagawa, and E. N. Olson Essential roles of the bHLH transcription factor Hrt2 in repression of atrial gene expression and maintenance of postnatal cardiac function PNAS, May 8, 2007; 104(19): 7975 - 7980. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
