Niessen Karsan 2007 Notch Signaling in The Developing Cardiovascular System
Niessen Karsan 2007 Notch Signaling in The Developing Cardiovascular System
Niessen Karsan 2007 Notch Signaling in The Developing Cardiovascular System
NOTCH PATHWAY (also known as RBP-J and CBF1) (123). The ankyrin repeats
are involved in protein-protein interactions including interac-
In mammals, the Notch receptor family consists of four type
tion with CSL; however, the seventh ankyrin repeat in coop-
I transmembrane receptors (Notch1 to Notch4) that regulate
eration with the TAD domain recruits transcriptional activators
cell fate decisions through cell-cell interaction (27). Notch
such as mastermind-like (MAML) and the histone acetyltrans-
receptors are translated as a large (⬃300 kDa) precursor
ferase (HAT) complex (69, 124). The PEST domain is in-
protein comprising an extracellular, a transmembrane, and an
volved in regulating protein half-life of the Notch receptors
intracellular domain that undergoes processing in the trans-
(31, 92).
Golgi network by Furin (76). Furin cleavage occurs at the
In mammals, there are five Notch ligands, Jagged1, Jagged2
S1 cleavage site and generates an extracellular fragment
(also called Serrate1 and Serrate2), Delta-like1 (Dll), Dll3, and
(NotchEC) and an extracellular-transmembrane-intracellular
Dll4, collectively referred to as the DSL (Delta/Serrate/Lag-2)
fragment (NotchTM) that is expressed on the cell surface as a
family (Fig. 1). DSL proteins are themselves type I transmem-
noncovalently linked heterodimer stabilized by a Ca2⫹ ion (5) brane proteins, with an extracellular domain comprised of 7–16
(Fig. 1). EGF-like repeats and a DSL domain, which is unique to Notch
The extracellular domain of Notch receptors is comprised of ligands. Jagged1 and Jagged2 have an additional cysteine-rich
29 –36 multiple epidermal growth factor (EGF)-like repeats, domain and a von Willebrand factor type C domain in the
depending on the specific Notch receptor, and 3 lin-12/Notch extracellular region (27).
(LNR) motifs. The EGF-like motifs are responsible for ligand The EGF-like repeats are thought to stabilize receptor-ligand
interaction, while LNR motifs are responsible for preventing interaction, while the DSL domain is responsible for Notch
receptor activation in the absence of receptor-ligand engage- receptor activation through interaction with EGF-like repeats
ment (35, 101, 104, 113). The intracellular domain of Notch 11 and 12 of the Notch receptors (104). The cysteine-rich
receptors is comprised of a recombination signal binding pro- domain of Jagged ligands are thought to control Notch receptor
tein-1 for J (RBP-J)-associated molecule (RAM) domain, binding specificity, while the von Willebrand factor type C
seven cdc10/ankyrin repeats, of which only the COOH-termi- domain is thought to be involved in ligand dimerization (27).
nal six assume the proper ankyrin fold, and a transactivation The intracellular regions of the DSL ligands are relatively short
domain (TAD) present in Notch1, Notch2, and Notch3. In and contain a PDZ domain in Jagged1 and Dll1 thought to be
addition, there are two nuclear localization signals, a glu- involved in activating downstream signaling through mecha-
tamine-rich stretch, and a PEST domain (2, 21, 69, 95, 139). nisms that are not well understood currently (1, 118).
The RAM domain is involved in potentiating Notch signaling
through interaction with the transcription factor CSL [C pro-
NOTCH RECEPTOR-LIGAND ACTIVATION
moter binding factor-1 (CBF1), suppressor of hairless, Lag-1]
Notch receptor-ligand interaction is thought to result in a
Address for reprint requests and other correspondence: A. Karsan, Dept. of
conformational change of the Notch extracellular domain ex-
Medical Biophysics, British Columbia Cancer Research Centre, 675 W. 10th posing a motif that is recognized and cleaved by the metallo-
Ave., Vancouver, BC, Canada V5Z 1L3 (e-mail: akarsan@bccrc.ca). protease tumor necrosis factor-␣ converting enzyme TACE/
http://www.ajpcell.org 0363-6143/07 $8.00 Copyright © 2007 the American Physiological Society C1
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Invited Review
C2 NOTCH AND CARDIAC DEVELOPMENT
Fig. 1. Notch receptors and ligands. There are 4 Notch receptors (Notch1–
Notch4) and 5 ligands [Jagged1/2, Delta-like (Dll)-1/3/4] in mammals. Notch
receptors are expressed on the cell surface as heterodimers stabilized through
calcium-dependent interactions. The extracellular domain contains 29 –36
epidermal growth factor (EGF)-like repeats (human Notch receptors), 3 Lin-
12/Notch (LNR) repeats, and a heterodimerization domain. The intracellular
domain contains an RBP-J-associated molecule (RAM) domain, 7 ankyrin
(ANK) repeats, 2 nuclear localization signals (NLS), a transactivation (TAD)
domain, and a PEST domain. Notch ligands are also expressed on the cell
surface. The extracellular domains contain a Delta/Serrate/Lag2 (DSL) domain
unique to Notch ligands and also contain multiple EGF repeats. Jagged1/2 also
contains a cysteine-rich domain and a von Willebrand factor type C domain.
The intracellular domains of Jagged1 and Dll1 have been shown to contain
PDZ domains, which may interact with downstream signaling components to
activate transcription. (For additional definitions of abbreviations in Figs. 1–3,
see text.)
migration of cardiac and anterior-cephalic mesoderm from the the embryo, the cardiac crescent fuses into a linear tube-like
primitive streak (61). Lineage analysis of the Mesp1-derived structure that starts beating at E8 in the mouse and at ⬃3 wk
cells demonstrated that the majority of cells in the myocardium of gestation in humans (117) (Fig. 3).
and endocardium are derived from Mesp1 expressing meso- In addition to the primary heart field, the existence of a
derm (111, 112). secondary heart field has been identified in both the developing
The endoderm underling the primary heart field plays an chick and mouse heart. The secondary heart field is located in
important role in specifying the cardiogenic phenotype. Using the splanchnic mesoderm that underlies the floor of the caudal
transplantation studies of mesoderm from a noncardiogenic pharynx and expresses many of the same markers as the
source transplanted into the cardiogenic field, Inagaki et al. primary heart field such as Nkx2.5 and GATA4, but also
(47) demonstrated that noncardiogenic mesoderm can be re- expresses unique makers such as FGF-10 and Nkx3.1 (60,
programmed to the cardiac fate. This reprogramming was 115). The extent of the contribution of cells from the secondary
further shown to be in part due to secreted factors from the heart field to the adult heart is not fully understood. In the
endoderm, such as members of the bone morphogenetic protein chick, the secondary heart field generates the smooth muscle
(BMP) family, sonic hedgehog, fibroblast growth factor cells of the conus and truncus only (14, 127). In the mouse,
(FGF)-8, and Crescent. In addition to the endoderm, the ecto- cells from the secondary heart field migrate into the arterial
derm secretes Wnt inhibitors that are required for induction of pole between E8.25 and E10.5, and, in addition to the smooth
a cardiogenic fate (reviewed in Ref. 6). muscle cells of the conus and truncus, a population of the
Even at this early stage in heart development, cell fate myocardial cells of the right ventricle are also generated from
analysis reveals that compartmentalization of heart chambers the secondary heart field (60) (Fig. 3, green).
has occurred. Myocardial atrial precursors are present in the
posterior region and ventricular progenitors in the anterior Heart Looping
region of the heart field (105). The most frequently used
Fusion of the cardiac crescent along the midline results in
primary heart field markers are Nkx2.5 (homolog of Drosoph-
the formation of the primitive heart tube. The primitive heart
ila Tinman), BMP2, Tbx20, GATA4, GATA5, and GATA6.
tube is composed of an outer myocardial cell layer and an inner
Although these markers are expressed in the primary heart
endocardial cell layer separated by a layer of extracellular
field, the exact boundaries of the primary heart field are not
matrix called the cardiac jelly. As heart development progresses,
precisely delineated by the expression of these genes. The heart
the linear heart undergoes several morphological changes that
field extends both laterally and medially of Nkx2.5 and BMP2
align and fuse the chambers of the heart. Mouse embryos that
expression, while BMP2 expression extends posterior and
are null for CSL, or Notch1-deficient embryos which also
anterior to the heart field (105).
express a Notch2-hypomorphic mutation, show randomized
The cardiac crescent is formed when the right and left heart
heart looping and axial rotation defects (65, 103). Additionally,
fields undergo anterior-medial migration and subsequently fuse
embryos deficient for the Notch ligand Dll1 display random-
at the anterior end (Fig. 3). Initially, all cells of the cardiac
ized heart looping and axial rotation (65, 97). However, this
crescent have cardiomyogenic potential, but signals from the
phenotype is not reproduced in Notch1-deficient or Notch2-
prospective myocardium and neurogenic tissue subdivide the
deficient embryos (38, 65). Defects in heart looping in Dll1-
cardiac crescent into ventral myogenic and dorsolateral non-
deficient embryos were found to be due to loss or misexpres-
myogenic domains (100). The ventral myogenic domain of the
sion of Nodal, Lefty2, and Pitx2, which are part of the evolu-
cardiac crescent gives rise to the myocardium of the heart tube,
tionarily conserved signaling cascade that controls left-right
while the dorsolateral nonmyogenic domain gives rise to the
morphogenesis. Krebs et al. (65) found that, in Dll1-deficient
mesocardial and pericardial roof cells (100). Studies in Xeno-
embryos, Nodal and Lefty2 expression was absent from the left
pus laevis have demonstrated that the Notch ligand Serrate1
lateral plate mesoderm (LPM), and Pitx2 expression was ran-
(Jagged1) but not Delta1 or Delta2 is expressed in the dorso-
domized in the LPM or not expressed at all (65). However,
lateral nonmyogenic domain, and the dorsal-most region of the
other investigators found that Nodal and Lefty2 expression was
myogenic domain expresses both Serrate1 and Notch1 in an
absent from only 50 and 25% of Dll1-deficient embryos,
overlapping pattern (107). Activation of the Notch pathway by
respectively. In the remaining embryos, Nodal and Lefty2 were
Serrate1 reinforces Serrate1 expression in the dorsolateral
expressed in the left, right, or both LPM (97). The specific
region and suppresses the myogenic potential of cells residing
reasons for the discrepancy between these two groups of
in this region (107). Although Notch activation suppresses
investigators remain to be elucidated, but is likely due to the
myogenic potential, expression of primary heart field markers
difference in genetic backgrounds of the two Dll1-deficient
Nkx2.5 and GATA4 is unaffected (107). In addition, CSL-
strains. Nodal expression was further shown to be dependent
deficient mouse embryos develop a primitive heart tube, and
on two CSL binding sites in a node-specific enhancer located
heart field specification appears normal (93, 119). These find-
⫺9.5 to ⫺8.7 kb 5⬘ of the nodal gene (65). In addition, ectopic
ings suggest that Notch signaling controls cardiac cell fate
activation of Notch signaling in early zebrafish embryos results
downstream of heart field specification.
in Nodal and Pitx2 expression in the right LPM and ultimately
Endocardial precursors are identifiable at the cardiac cres-
left-right patterning defects (103).
cent stage as a population of Flk1⫹/TAL1⫹-positive cells
distributed throughout the cardiac crescent (18). Lineage anal- Endothelial-to-Mesenchymal Transformation
ysis of Mesp1-derived mesoderm reveals that the endocardium
is derived from the same mesoderm as the myocardium, Beginning at E9.0 in the mouse, localized swellings of
thereby demonstrating that the endocardium and myocardium the cardiac jelly appear in the atrioventricular (AV) canal and
are of the same origin (111, 112). During subsequent folding of cardiac outflow tract (OFT), forming the superior and inferior
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Invited Review
NOTCH AND CARDIAC DEVELOPMENT C5
cardiac cushions. Cardiac cushions are acellular swellings of during early development (36). Notch1 activity is found to be
extracellular matrix protein secreted by the myocardium (22). highest in the trabecular endocardium, where it controls the
At E9.5, endocardial cells of the AV canal and OFT are expression of NRG1 and BMP10, which are responsible for
activated by signals emanating from the myocardium and by myocardial differentiation and proliferation, respectively (36).
interendocardial signaling pathways to undergo endothelial-to- In addition, constitutive expression of Notch1ICD in Mesp1-
mesenchymal transition. Endothelial-to-mesenchymal transi- derived cells results in abnormal heart morphogenesis (131). In
tion is a specific form of EMT required for mesenchymal cell Mesp1-derived cells, the Notch pathway is activated in the
formation from endocardial cells during cardiac cushion de- cardiogenic mesoderm and all subsequent cells of the endocar-
velopment. It is a critical process whereby endocardial cells dial and myocardial lineage (111, 112, 131). The most severe
undergo phenotypic and morphological alterations, resulting in defects induced by ectopic Notch activation in this model were
loss of apical-basolateral polarity and disruption of intercellu- defects in myocardial trabecular development, the appearance
lar junctions, and acquire the ability to degrade the basement of a cell mass in the AV canal, and right-shifted interventric-
membrane and migrate away from the confines of the endo- ular septum (131). Myocardial trabeculae were observed in the
thelial sheet to invade the underlying cardiac jelly. As devel-
compact myocardial layer and the AV canal region, two
opment progresses, the mesenchymal cells undergo prolifera-
regions where trabecular myocardium is usually absent (131).
tion, resulting in fusion of the cardiac cushions within the
In addition, in vitro experiments have clearly demonstrated that
lumen of the heart tube forming the initial septa. Further
remodeling of the cardiac cushion results in the formation of activation of the Notch pathway in endothelial cells results in
thin protruding leaflets comprised of endocardial cells and EMT (91). This study also showed that Notch-mediated EMT
extracellular matrix protein (ECM) that go on to develop into was cell autonomous and independent of TGF- signaling,
the heart valves. In the AV canal, EMT-derived cells are the supporting the importance of the role of Notch signaling during
sole contributor to the mitral and tricuspid valves, while in the EMT in heart development (91).
OFT, EMT- and neural crest-derived cells contribute to the The Notch target gene Hey2 is initially expressed in
aortic and pulmonary valves. ventricle precursors of the heart tube and later becomes
There are ongoing efforts to elucidate the transcriptional restricted to the compact myocardial layer of the ventricles.
networks operating during cardiac EMT (http://www. Hey2 is also highly expressed at the onset of EMT in the AV
mouseatlas.org/morgen/content). However, both loss- and canal endocardium (128). However, expression of Hey2 is
gain-of-function studies have revealed a critical role of the not sustained in the mesenchymal cells of the AV canal,
Notch pathway in regulating EMT during heart develop- suggesting Notch activity is required for initiation of EMT
ment. In the mouse, Notch1, Notch4, and Dll4 are expressed but not for maintaining a mesenchymal phenotype. Early in
in the AV canal and OFT endocardium at the onset of EMT. heart development, Hey1 is expressed in the lateral aspect of
Furthermore, both Notch1-deficient and CSL-deficient em- the cardiac crescent, which gives rise to the sinus venosus
bryos have significantly reduced EMT in the AV canal, as and atria. Later, Hey1 becomes restricted to the atrial
determined using an ex vivo AV canal explant assay, which myocardium. HeyL is not expressed during heart develop-
provides a measure of the degree of EMT taking place (125). ment, and levels of Hey1 and Hey2 in the heart gradually
Analysis of Notch1-deficient and CSL-deficient embryonic decline after birth. The mechanism by which Hey1 and
hearts reveals reduced expression of TGF-2 and its recep- Hey2 become differentially expressed remains to be re-
tors (125), which are critical initiators of EMT during heart solved, since both genes are direct targets of Notch activa-
development. However, by use of the AV canal explant tion. In the atrium, there is high expression of Jagged1,
assay, exogenous TGF-2 or TGF-3 was not sufficient to which could potentially regulate Hey1 expression (78, 126).
rescue the defect in EMT in Notch1-deficient and CSL- However, Notch receptors or ligands are not observed in
deficient embryos (125). These results suggest that, al- the compact myocardial layer, suggesting that, in this con-
though Notch signaling is important for expression of text, Hey2 may be regulated by a pathway other than Notch
TGF-2 pathway components, it is not clear whether Notch (126).
is downstream of, upstream of, or synergistically required
Several studies have also revealed a critical role of Hey2
for TGF--dependent EMT during heart development A
during heart development. The phenotypes of Hey2-null
caveat of interpreting data from the Notch1 and CSL mutant
embryos is the significant delay in embryonic development mice are variable, but these mice have high mortality in the
observed in these mutants. The delay is particularly apparent first weeks after birth because of cardiovascular defects,
in the heart, and therefore EMT might be significantly including ventricular septal defects, pulmonic stenosis, AV
delayed but not impaired. In fact, in AV canal explant canal valve irregularities, and cardiac hypertrophy (17, 25,
assays, a very small population of ␣-smooth muscle actin 63). Gene targeting studies have revealed that Hey1 or HeyL
(SMA)-positive cells is detectable, suggesting that Notch1- alone is not required for heart development. However, Hey1
deficient and CSL-deficient embryos are capable of initiat- and Hey2 double-deficient embryos die at E9.5 because of
ing cardiac EMT at a reduced level (125). Supporting a role severe heart defects, including missing ventricular trabecu-
for the Notch pathway in EMT of the AV canal, it was lae and lack of arterial differentiation (25). In addition,
shown that zebrafish injected with constitutively active combined loss of both Hey1 and HeyL results in a signifi-
Notch1 developed hypercellular AV canals and enlarged AV cant reduction in endocardial EMT, similar to the Notch1-
valves as a result of increased EMT (125). or CSL-deficient embryos (26). Known expression patterns
Notch1-deficient embryos also display defects in ventricular of Notch pathway component have been summarized in
trabeculation because of a decrease in myocardial proliferation Table 1.
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Invited Review
C6 NOTCH AND CARDIAC DEVELOPMENT
Notch1 Expressed in the cardiac crescent (E7.5). At E8.0 to E11.5 expression is limited to the entire endocardium and highly expressed
in the AV canal and outflow tract endocardium (124, 132).
Notch2 Expressed in the AV canal endocardium (E12.5) and the outflow tract (E11.5 and 14.5). Expressed in atrial and ventricular
myocardium (E13.5) (76, 80, 82).
Notch3 Expressed in the cardiac crescent (E7.5) but not detected after heart tube formation (E8.0) (132).
Notch4 Expressed in the endocardium (E10.5) (90).
Jagged1 Expressed in the AV canal and outflow tract endocardium and atrial myocardium (E10.5–E12.5) (77).
Jagged2 Expression has not been analyzed
DI11 Not expressed in the heart (4)
DI13 Expression has not been analyzed
DI14 Expressed in the cardiac crescent (E8.0) and the endocardium from E8.5 onward. Expression is further restricted to the
ventricular endocardium after E11.5 (3, 19).
Hey1 Expressed in the lateral portion of the heart tube (E8.5) and the endocardium and septum transversum (E9.5). Expressed
exclusively in the atrial myocardium at E10.5 (71, 124).
Hey2 Expressed in the anterior portion of the heart tube (E8.5) and the AV canal and OFT endocardium (E11.0). Highly expressed in
the subcompact ventricular myocardium (E10.5) (12, 71).
HeyL It has been reported that HeyL is not expressed in the embryonic heart (72), but combined targeting of Hey1 and HeyL results
in cardiovascular defects related to EMT (26).
AV, atrioventricular; E, embryonic day; OFT, outflow track; EMT, epithelial-to-mesenchymal transition.
ARTERIAL VENOUS SPECIFICATION disorganized, and the dorsal aorta and cardinal vein are formed
but are significantly reduced in size (67). These results suggest
Early Vascular Progenitors
that Notch1 is not required for angioblast differentiation but is
Vasculogenesis is the de novo development of endothelial required for remodeling of the vascular plexus. Notch4-defi-
cells from mesodermal progenitors called angioblasts, while cient mice are viable and fertile; however, Notch1/Notch4
angiogenesis is the formation of new vessels from preexisting double-deficient mice have vascular defects similar to but more
vessels. All embryonic mesoderm, with the exception of the severe than Notch1-deficient animals (67). This suggests a
prechordal plate, has angioblast potential (89). Angioblast partial redundancy between Notch1 and Notch4 in the devel-
differentiation in situ forms a primary vascular plexus that oping vasculature. Notch2-deficient mice die at E11.5 because
undergoes extensive remodeling into arteries, veins, and cap- of a variety of defects in the cardiovascular and renal systems
illaries. The three types of vascular structures are morpholog- (38, 82). Notch3-deficient mice, in contrast, are viable without
ically, functionally, and molecularly distinct. At the molecular any obvious defects in cardiovascular development (68). Vas-
level, artery-specific factors include Ephrin-B2, CD44, Neuro- cular remodeling defects are also seen in Dll4- and Jagged1-
pilin-1, Hey1, and Hey2, while vein-specific factors include deficient mice, resulting in death at E9.5 and E11.5, respec-
EphB4, COUP-TFII, and Lefty1 (11, 25, 135). tively (32, 66, 134). Expression of constitutively active Notch4
driven by the flk1 promoter in endothelial cells during devel-
Expression of Notch Pathway Components opment results in yolk sac remodeling defects and disorganized
Notch pathway components are initially expressed through- embryonic vessel development.
out the developing cardiovascular system but become mainly
restricted to cells that will acquire an arterial fate later in Molecular Patterning of Arterial Venous Specification
development. At E9.5, Notch1, Notch4, and Dll4 are expressed Arterial and venous endothelial progenitors have been
throughout the primitive vascular plexus. By E13.5, Notch1, shown to be specified before vessel assembly or blood flow
Notch2, Notch4, Dll4, Jagged1, and Jagged2 expression has (41). Recent studies have revealed a signaling cascade for
become mainly restricted to the arterial endothelium (126). arterial specification that involves sequential activation of
Notch1 and Dll4 are also expressed in the capillary endothe- Sonic Hedgehog (Shh), vascular endothelial growth factor
lium. In contrast, artery smooth muscle cells express low levels (VEGF), and Notch (71). In zebrafish, Shh deficiency results in
of Notch1 and high levels of Notch3 and Jagged1 (126). a loss of arterial specification and decreased VEGF expression
Notch4 is also expressed in the cardinal vein, human umbilical (71). In contrast, Shh activation in zebrafish results in upregu-
endothelial cells, and capillary endothelium. Expression pat- lation of VEGF and ectopic arterial specification (as deter-
terns of Notch receptors and ligands in the vasculature have mined by expression of EphrinB2) of vessels otherwise des-
been extensively reviewed in Ref. 49. When injured, arteries tined to become veins (71). Activation of the Notch pathway in
induce expression of various Notch receptors and ligands (75). mature vessel endothelium, via Tie2-driven inducible expres-
Defects in Vascular Remodeling sion of Notch4ICD, results in expression of EphrinB2 and
increased smooth muscle layers resulting in arterialization
Notch1-deficient mice die before E10.5 because of severe of the venous vessels (8). In this model, overexpression of
defects in cardiovascular development (122). In Notch1-defi- Notch4ICD results in death of adult animals within weeks of
cient embryos, the yolk sac and cerebral primary vascular expression. Interestingly, the effects of Notch4ICD expression
plexus are formed, but subsequent remodeling of the vascular are reversible on reversal of Notch4ICD expression (8). In
plexus is defective. In addition, intersomitic vessels are highly mice, Hey1/ Hey2 or Notch1/Notch4 double-deficient and
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Invited Review
NOTCH AND CARDIAC DEVELOPMENT C7
Dll4-deficient embryos display defects in arterial specification above, Notch pathway components are highly expressed in
to various degrees (25, 67). Studies in zebrafish have provided arterial endothelial cells, which correlates with the importance
significant insight into the role of the Notch pathway in of the Notch pathway in regulating endothelial function in the
arterial-venous specification. Enforced expression of Notch5, aortic valve. With the use of an in vitro system, it was
which is similar to mammalian Notch3 by sequence, in the demonstrated that Notch1, Hey1, and Hey2 repress the func-
endothelium results in reduced venous expression of flt-4, a tion of the transcription factor RUNX2 (33). RUNX2 has been
venous marker (70). Conversely, blocking Notch signaling in linked to valvular calcification in both rabbit and mouse, where
the dorsal aorta, using dominant-negative CSL, results in loss it regulates expression of several osteogenic genes, such as
of arterial expression of EphrinB2 (70). osteopontin and osteocalcin (20). It was further suggested that
Notch target genes have also been shown to play a role in Notch signaling via upregulation of Hey1 and Hey2 results in
arterial-venous specification. A mutant of gridlock, the only Hey1 or Hey2 physically interacting with RUNX2, thereby
Hey family member expressed in the zebrafish vasculature, inhibiting RUNX2 function (33). It is not known whether
shows defects in aortic assembly resembling coarctation of the Notch1 mutations found in humans result in lower Hey1 and
aorta (137). Antisense-directed knockdown of gridlock sup- Hey2 expression in the aortic valve, thereby allowing higher
presses arterial marker expression and expands contiguous RUNX2 activity, subsequent expression of osteogenic genes,
regions of the vein during embryonic vascular development and calcification of the aortic valve.
(136). However, overexpression of gridlock acts to repress
venous fate rather than promote arterial specification (136). Alagille Syndrome
The role of gridlock is controversial, as others have suggested
Mutations in the Jagged1 locus are associated with 94% of
that Notch mediates arterial specification independently of
patients with Alagille syndrome (AGS) (130). In addition, muta-
gridlock (70). In addition, VEGF stimulation was shown to
tions in the Notch2 locus have been identified in patients with
upregulate Notch1 and Dll4 expression exclusively in arterial
Jagged1-independent AGS (84). AGS is an autosomal dominant
endothelial cells. VEGF treatment of Shh-deficient but not
disorder most commonly associated with neonatal jaundice and
Notch-deficient embryos is capable of rescuing arterial
impaired development of intrahepatic bile ducts, with additional
EphrinB2 expression.
abnormalities of the eye, heart, kidney, and skeleton with variable
In venous vessels, expression of the orphan nuclear receptor
penetrance. The most common cardiovascular defect is peripheral
COUP-TFII was shown to regulate the expression of Notch1
pulmonic stenosis, and 13% of AGS patients have Tetralogy of
and Jagged1 in arterial-venous specification (135). Loss of
Fallot (58, 64, 85). Tetralogy of Fallot is a condition characterized
COUP-TFII resulted in ectopic venous expression of Notch1
by ventricular septal defect, overriding aorta, infundibular pulmo-
and Jagged1, resulting in arterialization of the venous vessels
nary stenosis, and often right ventricular hypertrophy. In less
(135). In contrast, ectopic expression of COUP-TFII in the
frequent cases, AGS has been associated with other defects of the
endothelium, via the Tie2 promoter, resulted in reduced
cardiac cushion (23, 85). The cardiac AGS phenotype is consis-
Jagged1 expression and arterial expression of EphB4, a venous
tent with the expression pattern of Jagged1 and Notch receptors in
marker. Together these findings suggest that COUP-TFII is
the cardiovascular system. Analyses of mutations in the Jagged1
upstream of Notch signaling in arterial-venous specification,
locus have revealed in some cases complete loss of the Jagged1
and that Notch signaling represses venous differentiation. Thus
locus and in other cases inactivating mutations that lead to a
the current paradigm depicts a signaling cascade for arterial
misexpressed or truncated Jagged1 protein (120). However, het-
differentiation where Shh is upstream of VEGF and VEGF is
erozygous mutations in Jagged1 or Notch2 do not reproduce the
upstream of Notch signaling.
AGS phenotype in mice (38, 134). Jagged1 heterozygote mice
MUTATIONS OF NOTCH PATHWAY COMPONENTS IN display eye defects, while Jagged1-null mice die at E10 because
HUMAN DISEASE of vascular defects (134). Notch2 haploinsufficiency in mice
results in kidney defects and myocardial hypoplasia, but the
Aortic Valve Disease phenotype does not resemble findings of cardiovascular and
kidney defects in patients with AGS (38, 81). However, mice
In humans, mutations in the Notch1 locus result in a spec-
doubly heterozygous for a Jagged1-null and Notch2-hypomorphic
trum of heart defects (33). The most prevalent malformations
allele develop jaundice and impaired development of intrahepatic
are bicuspid aortic valve disease and calcification of the aortic
bile ducts with associated abnormalities of the eye, heart, and
valve (33). Calcification of the aortic valve is the third leading
kidney, reproducing an AGS phenotype (83). The reason that
cause of heart disease in adults, while the presence of the
human Jagged1 haploinsufficiency results in AGS while in mice
bicuspid aortic valve is present in 1–2% of the population (43).
there is an additional requirement for Notch2 insufficiency is
Mutations in the Notch1 locus result in a premature stop codon
unknown. Possible explanations include a higher basal expression
in the extracellular domain of Notch1, likely resulting in rapid
of Jagged1 in mice, compensating expression of a Notch ligand,
degradation of mRNA by the nonsense-mediated mRNA decay
or increased avidity of Notch receptor-ligand interaction in the
pathway (33). The resulting defects may be due to halploin-
mouse cardiovascular system.
sufficiency, or alternatively, the premature stop codon could
result in the expression of a truncated Notch1 protein that could Cerebral Autosomal Dominant Arteriopathy with Subcortical
function as a dominant-negative mutant. These hypotheses Infarcts and Leukoencephalopathy
have yet to be tested experimentally. The mechanism by which
human Notch1 mutations effect aortic valve calcification is Cerebral autosomal dominant arteriopathy with subcortical
poorly understood. However, calcification of the aortic valve is infarcts and leukoencephalopathy (CADASIL) is an autosomal
thought to be due to endothelial dysfunction. As discussed dominant disorder associated with defects in arterial vascular
AJP-Cell Physiol • VOL 293 • JULY 2007 • www.ajpcell.org
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Invited Review
C8 NOTCH AND CARDIAC DEVELOPMENT
homeostasis, resulting in increased incidence of stroke, mi- signaling in the vasculature. However, a thorough understand-
graine headaches, and mood disturbances (10, 55). CADASIL ing of the downstream targets of Hey proteins and the function
results from a progressive loss of the arterial vascular smooth of these downstream targets remains to be elucidated. Early
muscle cells and increased vascular fibrosis, leading to narrow- studies on functional changes triggered by Notch in vascular
ing of the lumen of small and medium arteries (10). CADASIL cells have been performed, but much remains to be investi-
is histologically characterized by accumulation of granular gated in this area also. Notch signaling appears to integrate
osmophilic deposits (GOMs) in the vessel media (80, 108). with feedback loops of the VEGF, platelet-derived growth
Arterial vascular defects seen in CADASIL patients can be factor, Wnt, and BMP pathways, but the molecular mecha-
systemically found; however, for unknown reasons, vascular nisms of how this occurs are still under investigation. Undoubt-
complications are only found in the brain (110). edly, there will be further examples of vascular diseases that
Missense point mutations in the first five EGF repeats in the are influenced or perhaps even caused by aberrant signaling in
Notch3 locus resulting in an odd number of cysteine residues Notch pathway genes as investigators place greater emphasis in
in the EGF repeat regions are found in 95% of CADASIL cases these unmined areas of research.
(16, 54, 56). The change in the number of cysteine residues
from six to five or seven in the EGF repeats is thought to result GRANTS
in a conformational change of the EGF repeats, leading to Work in the laboratory of A. Karsan is supported by grants from the Heart
accumulation of the ectodomain of the Notch3 receptor. As and Stroke Foundation of British Columbia and the Yukon, the Canadian
discussed above, Notch receptors are expressed as a het- Institutes of Health Research, the Canadian Cancer Society, the Stem Cell
Network, Genome Canada, and Genome British Columbia. K. Niessen was
erodimer on the plasma membrane. In patients with CADASIL, supported by a Doctoral Research Award from the Michael Smith Foundation
there is a specific accumulation of the 210-kDa ectodomain of for Health Research. A. Karsan is a Senior Scholar of the Michael Smith
Notch3 in the vascular smooth muscle plasma membrane in Foundation for Health Research.
close association with but not within the GOMs (53).
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