Download Endocardial Notch Signaling in Cardiac Development and Disease

Review
Endocardial Notch Signaling in Cardiac
Development and Disease
Guillermo Luxán,* Gaetano D’Amato,* Donal MacGrogan,* José Luis de la Pompa
Abstract: The Notch signaling pathway is an ancient and highly conserved signaling pathway that controls cell
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fate specification and tissue patterning in the embryo and in the adult. Region-specific endocardial Notch activity
regulates heart morphogenesis through the interaction with multiple myocardial-, epicardial-, and neural crest–
derived signals. Mutations in NOTCH signaling elements cause congenital heart disease in humans and mice,
demonstrating its essential role in cardiac development. Studies in model systems have provided mechanistic
understanding of Notch function in cardiac development, congenital heart disease, and heart regeneration.
Notch patterns the embryonic endocardium into prospective territories for valve and chamber formation,
and later regulates the signaling processes leading to outflow tract and valve morphogenesis and ventricular
trabeculae compaction. Alterations in NOTCH signaling in the endocardium result in congenital structural
malformations that can lead to disease in the neonate and adult heart. (Circ Res. 2016;118:e1-e18. DOI: 10.1161/
CIRCRESAHA.115.305350.)
Key Words: cardiomyopathies
■
endocardium
■
heart valves
T
he assembly and function of a complex organ such as the
heart requires the precise coordination of multiple distinct tissues. The endocardium is a specialized endothelial tissue lining the interior of the heart, closely juxtaposed to the
myocardium and in direct continuity with the vascular endothelium. Among the 3 tissue layers forming the mature adult
heart (endocardium, myocardium, and epicardium), comparatively less attention has been paid to the endocardium, as it
was primarily thought as a physical barrier serving to regulate
vascular permeability between blood and subjacent cardiac
muscle. However, the importance of the endocardium as a
crucial regulator of adult cardiac function derives essentially
from its ability to adjust heart pumping to hemodynamic and
hormonal demands by modulating cardiac muscle contractile
performance and rhythmicity.1 Moreover, recent data demonstrate that the mechanical properties of the aortic valve cusps
(ie, stiffness) are regulated by the endothelium.2
The endocardium also plays crucial roles during heart development because it provides patterning signals for myocardial trabeculation3 and compaction,4 contributes to coronary
vessel formation,5 and in conjunction with regionalized signals from the myocardium, cells from specific regions of the
endocardium will undergo epithelial–mesenchymal transition
(EMT) to form the endocardial cushions (ECs). The EC will
form several important structures within the heart, including
■
heart ventricles
■
Notch signaling
the valve leaflets and the membranous walls of the ventricular
and atrial chambers.6,7 In addition, endothelial-derived cells
and cells of neural crest origin function in the EC to separate
the common outflow tract (OFT) into the pulmonary artery
and aorta.8 In keeping with these multiple requirements, it
is evident that localized signaling cross talk must take place
early to establish a tight functional coupling between the endocardium and the myocardium and ensure normal cardiac
growth and homeostasis throughout life.
In this review, we focus on the role of Notch in the endocardium in the context of signaling interactions with the other
cardiac tissues to coordinate heart morphogenesis. First, we
explain briefly the elements of the Notch signaling pathway,
their expression during cardiac development, and the endocardial (and coronary endothelial) activity of the pathway.
Second, we describe the developmental origins of the endocardium and its relationship to cardiac and other endothelial
lineages, and go on to examine key cardiogenic processes that
require cell autonomous and non–cell-autonomous endocardial Notch signaling: patterning of the early embryonic endocardium into prospective territories for valves and ventricular
chambers; and the later processes of trabecular compaction
and morphogenesis of the OFT and valves. Finally, we provide examples of how Notch dysfunction in the endocardium
results in cardiac structural malformations that can lead to
Original received July 28, 2015; revision received October 16, 2015; accepted October 22, 2015.
From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovascular (CNIC),
Melchor Fernández Almagro, Madrid, Spain (G.L., G.D’A., D.M., J.L.d.l.P.); and Department of Tissue Morphogenesis, Max Planck Institute for Molecular
Biomedicine, Münster, Germany (G.L.).
*These authors contributed equally to this article.
Correspondence to José Luis de la Pompa, PhD, Intercellular Signaling in Cardiovascular Development and Disease Laboratory, Centro Nacional de
Investigaciones Cardiovascular (CNIC), Melchor Fernández Almagro 3, E-28029 Madrid, Spain. E-mail [email protected]
© 2016 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.115.305350
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Nonstandard Abbreviations and Acronyms
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AAA
AGS
AVC
BAV
CHD
CNC
EC
EMT
FHF
GOF
LOF
LV
LVNC
NICD
OFT
SHF
SLV
VSD
WT
aortic arch arteries
Alagille syndrome
atrioventricular canal
bicuspid aortic valve
congenital heart disease
cardiac neural crest
endocardial cushion
epithelial–mesenchymal transition
first heart field
gain-of-function
loss-of-function
left ventricle
left ventricular noncompaction
Notch intracellular domain
outflow tract
second heart field
semilunar valve
ventricular septal defect
wild-type
disease in the neonates and adults, and speculate about the
signaling and structural function played by the endocardium
in the regenerating zebrafish heart.
Notch Pathway Elements
Notch is an evolutionary conserved signaling pathway that
regulates cell fate specification, differentiation, and tissue patterning both in development and adulthood.9 Notch proteins
are single-pass transmembrane receptors with a large extracellular domain composed of a variable number of tandem
epidermal growth factor–like repeats, followed by a shorter
membrane-spanning portion and an intracellular domain
(NICD), which contains a transcriptional activation domain
(Figure 1). Notch proteins are processed in the Golgi by
proteolytic cleavage by a furin-like convertase10 (Figure 1,
S1 cleavage,), whereas sugars are added to the epidermal
growth factor–like repeats of Notch extracellular domain by
various glycosyl transferases, including those of the Fringe
family (Figure 1). Modified Notch is then targeted to the
cell surface as a heterodimer held together by noncovalent
interactions. Once in the membrane, the Notch extracellular domain is available to interact with membrane-bound ligands of the Delta (Dll1-4 in mammals) or Jagged (Jag1 and
Jag2) families expressed by neighboring cells, and cell–cell
contact is required for signaling (Figure 1). Whether Notch
ligand–receptor interactions occur through monomeric or dimeric membrane complexes remains the subject of intense
debate.11–13 Productive ligand–receptor interaction depends on
the activity in the signaling cell of E3 ubiquitin ligases such
as mind bomb-1 (Mib1), which ubiquitylates the ligand in its
cytoplasmic C-terminal tail, an event crucial for its endocytosis and effective Notch signaling.14 Ligand endocytosis generates mechanical force to pull on Notch,15,16 which in turn
induces conformational changes leading to exposure of the
S2 site of the receptor recognized by ADAM (A Disintegrin
And Metalloprotease-containing) proteins (Figure 1).17,18 The
remaining Notch fragment becomes susceptible to cleavage
by γ-secretase (at the S3 site)19,20 resulting in release of NICD,
which translocates to the nucleus of the signal-receiving cell.13
When released from the cell membrane, NICD can function
as a transcription factor.21 In the nucleus, NICD binds directly to the DNA-binding protein CSL (CBF1, Suppressor
of Hairless, Lag-1; Figure 1).22 NICD–CSL binding displaces
corepressors and histone deacetylases that repress target genes
in the absence of Notch signaling and allows recruitment of
the transcriptional coactivator mastermind-like. Formation of
the CSL–NICD–mastermind-like ternary complex allows recruitment of additional coactivators to activate transcription
of target genes, typically those encoding repressors such as
basic-helix-loop-helix transcription factors of the Hes and
Hey families.23 Nevertheless, the spectrum of direct Notch
targets is large and tissue specific, including Snail1,24,25 p21,26
c-Myc,27 EphrinB2,3 and Nrarp28 (Figure 1).
Cardiac Development and Endocardial Notch
Activity
The heart develops in the mouse embryo at around E7.5 from
bilateral precardiac mesoderm cells that form the cardiac crescent (Figure 2A).8 The crescent contains 2 populations of precardiac cells: the first and second heart fields (FHF and SHF;
Figure 2A) that include progenitors of the first cardiac tissues,
the myocardium and endocardium.29 The crescent fuses at the
midline forming a primitive heart tube consisting of an inner
endocardial layer and an outer myocardial layer separated by
an extracellular matrix termed cardiac jelly (Figure 2A and
2B). The heart tube grows at both ends by addition of SHF progenitors into its anterior (arterial) and posterior (venous) poles
and simultaneously undergoes rightward looping morphogenesis30 (Figure 2C and 2D), changing the initial anterior–posterior polarity into right–left patterning. The FHF will give rise
to the left ventricle (LV) and other parts of the heart except
the OFT, whereas the SHF gives rise to the OFT myocardium
and other parts of the heart except for the LV (Figure 2C and
2D). At E9.5, uneven growth within the heart tube results in
ballooning of the chambers and establishment of the trabecular network31 (Figure 2D). The endocardium lines the lumen
of the cardiac chambers, and through an EMT process forms
the cardiac cushion mesenchyme in the atrioventricular canal
(AVC) and proximal OFT (Figure 2D). At E9.5, a third tissue
layer covering the myocardium develops from the proepicardium, a mass of coelomic progenitors located at the venous
pole of the embryonic heart (Figure 2D). Proepicardium
cells attach to and spread over the myocardium to form the
primitive epicardial epithelium. The epicardium gives rise to
a population of epicardium-derived cells through EMT, which
invade the heart and differentiate into various cell types (see
below). From E10.5 onward, the OFT is remodeled from a
single OFT vessel, which circulates blood into the 3 main aortic arch arteries (AAAs) to join 2 dorsal aortae that distribute the blood throughout the embryo. Neural crest emanating
from the dorsal neural tube invades the AAAs to reach the
OFT (Figure 2E) and differentiates into smooth muscle cells
Luxán et al Endocardial Notch Signaling e3
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Figure 1. The Notch signaling pathway. In the signaling cell, membrane-bound Notch ligands (Dll1, 3, 4, and Jag1, 2) are characterized by
a Delta/Serrate/Lag2 motif (light yellow) located in the extracellular domain. Ligand activity is regulated by the ubiquitin ligase Mind bomb-1
(Mib1) through ubiquitinylation of the intracellular domain (dark yellow squares). In the signal-receiving cell, the Notch receptor (Notch 1–4)
is processed at the S1 site by a furin protease, sugar-modified by Fringe in the Golgi and is thought to be inserted into the membrane as
a heterodimer with a large extracellular domain (NECD). Ligand–receptor interaction leads to 2 consecutive cleavage events (at S2 and S3
sites, respectively) performed by an ADAM (A Disintegrin And Metalloprotease-containing) protease and presenilin, which release the Notch
intracellular domain (NICD) that translocates to the nucleus and binds to CSL (CBF1, Suppressor of Hairless, Lag-1). In the absence of NICD,
CSL associates with corepressor proteins (Co-R) and histone deacetylases to repress transcription. After NICD binds to CSL, conformational
changes occurring in CSL displace transcriptional repressors. The transcriptional coactivator Mastermind (MAML) is then able to bind to
NICD/CSL to form a ternary complex that recruits additional coactivators (Co-A) to activate transcription of a set of target genes.
covering the AAAs endothelium (Figure 2E). Progressively,
the AAAs remodel to form the mature aortic arch and major branches (Figure 2E). Neural crest cells also invade the
OFT and contribute mesenchyme to the OFT septum and SLV
cusps (Figure 2F). In the mature 4-chambered heart, the myocardium forms the contractile tissue of the ventricular walls,
the epicardium contributes to a subset of coronary endothelial
cells, coronary smooth muscle cells, part of the AV valves, and
the cardiac fibroblasts, and the endocardium gives rise to the
cardiac valves and coronary vessel endothelium (Figure 2F
and 2G). Defective endocardial Notch signaling affects cardiac valve and chamber development, leading to diseases
such as bicuspid aortic valve (BAV) and LV noncompaction
(LVNC) cardiomyopathy (Figure 2H and 2I). Notch signaling is also involved in epicardium and coronary vessel formation,32,33 a topic beyond the scope of this review that has been
comprehensively discussed.34–36
The Notch pathway is active early in cardiac development.37 At E9.5, the Jag1 ligand is expressed in the endocardium delineating the presumptive valve territory of the AVC
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Figure 2. Key events in cardiac development. A, At human embryonic day 14 (hE14) or mouse E7.0. In the gastrulating embryo
cardiac progenitors (purple) migrate into the anterior half of the primitive streak to reach the head folds. At E7.5, progenitor cell populations
of the first and second heart fields (FHF, purple; SHF, yellow) have fused in the midline of the embryo. At E8.0, the heart tube stage, the
approximate contributions of FHF and SHF are shown. B, The heart tube has an outer myocardial layer (light brown) and an inner endocardial
endothelium (red) separated by cardiac jelly (gray). C and D, At E9.0, the heart is elongated and looping rightward (C). At E9.5, formation of
the atrioventricular canal (AVC) separates the 2 chambers’ territories, the developing atria and ventricles (C). Formation of the valve primordia
occurs around E10, together with ventricular chamber development that begins with trabeculae formation in the developing left and right
ventricles, and epicardial formation from the proepicardium, located in the venous pole of the heart (D). E, Outflow tract (OFT) and aortic arch
arteries (AAA) remodeling is dependent on the cardiac neural crest (CNC) cells. The OFT region of the heart initially exists as a single outflow
vessel, which circulates blood into three major pairs of AAAs to join 2 paired dorsal aortae that distribute the blood throughout the embryo.
Left, Neural crest cells migrate from the dorsal neural tube, surrounding the aortic arch arteries to the cardiac outflow (Continued )
Luxán et al Endocardial Notch Signaling e5
Figure 2. Continued tract. Middle, The neural crest cells that invest the endothelial tubes of the AAAs differentiate into vascular
smooth muscle cells. Right, the AAAs remodel to form the mature aortic arch, with neural crest–derived vascular smooth muscle cells
contributing to most of the aortic arch and its major branches. F, Contribution of neural crest (purple) and epicardial (blue)–derived cells
(NCDC and EPDC) to the cardiac valves. NCDCs migrate through the pharyngeal arches and into the OFT to initiate the reorganization
of the OFT and formation of semilunar valves. EPDCs contribute to the formation of the coronary arteries, the interstitial cells in the
myocardium, and the AV valves. In a top view of the septated heart, the relative contributions of NCDCs and EPDCs to cardiac valve
leaflets and cusps are shown. G, E13.5-birth. The 4 chambers and valves are shown. The epicardium is shown in gray, and the coronary
vessels in red and blue. H, Cushion fusion between E11.5 and E12.5 is a critical step in valve morphogenesis. Aberrant fusion events can
lead to bicuspid aortic valve (BAV). R–NC and R–LC fusions might have different causes. I, Scheme depicting the thick ventricular walls of
the normal adult heart and the thin and trabeculated walls of a noncompacted heart. In all panels, the ventral aspect of the heart is shown
(anterior to the top). A indicates atria; la, left atrium, lv, left ventricle; LVNC, left ventricular noncompaction; mv, mitral valve; pv, pulmonary
valve; rv, right ventricle; and tv, tricuspid valve.
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and throughout the myocardium (Figure 3A–3A″ and 3D),38
whereas Dll4 is expressed in the endocardium (Figure 3B–
3B″ and 3D).3 Mib1 is expressed in both endocardium and
myocardium4 and has the potential to ubiquitylate both ligands. At this stage, Notch1 activity is relatively uniform in
the AVC and the OFT endocardium but restricted to the endocardium at the base of the developing trabeculae in the ventricles (Figure 3C–3C″ and 3D).37 At E12.5, Jag1 is expressed
in the myocardium around the semilunar valves (SLVs) and
in the endocardium (Figure 3E and 3H) where Notch is active
(Figure 3G and 3H). Dll4 expression in valve endocardium
is greatly diminished when compared with E9.5 (Figure 3F
and 3H). In the chambers, myocardial Jag1 expression is
maintained (Figure 3I and 3L), Dll4 expression is attenuated
(Figure 3J and 3L), and Notch activity remains widespread
in the endocardium (Figure 3K and 3L).4 Once the general
cardiac pattern is established, substantial cellular proliferation, tissue morphogenesis, and terminal differentiation of
the different cardiac structures occur, and the heart becomes
fully functional at birth (Figure 2G). In late gestation, Jag1
Figure 3. Endocardial Notch activity during cardiac development. A–D, In the E9.5 heart, the ligands Jag1 and Dll4 are expressed in
the atrioventricular canal (AVC) endocardium (A, A′, B, B′, and D) and in chamber myocardium and endocardium, respectively (A, A″, B,
B″, and D), whereas the Notch1 receptor is active in the endocardium (C–C″, and D). E–H, At E12.5, Jag1 is strongly expressed in the
endocardium lining the outflow tract endocardial cushions (E and H), whereas Dll4 expression is reduced when compared with E9.5 (F
and H), and endocardial N1ICD expression persists (G and H). I and L, At E12.5, Jag1 is strongly expressed in trabecular myocardium (I,
arrowhead, L) and weakly in the compact myocardium, whereas Dll4 is weakly expressed in endocardium (J, arrowhead, L) and N1ICD
is expressed throughout chamber endocardium (K, arrowhead, L). M–P, At E14.5, Jag1 is expressed in trabecular myocardium (M,
arrowhead, P), Dll4 in the coronary vessels (N and P), and N1ICD in chamber endocardium and coronary vessels endothelium (O and P). a
indicates atria; avc, atrioventricular canal; cv, cardiovascular; la, left atrium, lv, left ventricle; and rv, right ventricle.
e6 Circulation Research January 8, 2016
is expressed in smooth muscle tissue around the valves (not
shown), and in chamber myocardium (Figure 3M and 3P),
Dll4 in developing coronary vessels (Figure 3N and 3P)4
and Notch1 activity persists in valve and chamber endocardium (Figure 3O and 3P). Defective NOTCH signaling has
been shown to cause various cardiac pathologies including
Alagille syndrome (AGS),39,40 BAV41 (Figure 2H), and LVNC4
(Figure 2I). We suggest that these pathologies result from the
disruption of the NOTCH-mediated communication between
the endocardium and the surrounding cardiac tissues.
Origin of the Endocardium
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The timing of endocardial specification and whether the endocardium is derived from progenitors that lack myocardial
potential is controversial.42 Data from chick and zebrafish
models involving retroviral single-cell tagging and tracking
studies suggest that specification of endocardial and myocardial cells occurs before their ingression through the primitive
streak during gastrulation, perhaps as early as the blastula
stage and significantly before their migration to the heart
field.43,44 Another prevailing view inferred mainly from mouse,
embryonic stem cell models, and cell lineage-mapping studies
contended that endocardial and myocardial cells in mammals
are derived from a common multipotent progenitor within the
cardiac mesoderm still present at the cardiac crescent stage.45
Moreover, although it was clear that the OFT endocardium
and myocardium both derived from a common Flk1+ precursor distinct from endothelial cells46 and therefore crediting the
existence of a SHF bipotential progenitor, another study suggested that the OFT endocardium is derived at least in part
from Flk1+ vascular endothelial cells lacking myocardial potential,47 implying that the SHF contains distinct myocardial
and endothelial/endocardial lineages. A recent cell fate clonal
analysis satisfactorily reconciles these apparently opposing
sets of data. Using multicolor inducible Mesp1 reporter constructs, it was found that Mesp1 marks distinct sets of cardiac
progenitor cells with progressively restricted lineage differentiation at different time points during gastrulation.48 Mesp1
progenitors give rise to the FHF at the early primitive streak
stage (E6.25) and then to the SHF progenitors at late primitive
streak stage (E7.25) with the expression of Mesp1 in the 2
populations overlapping at E6.75. FHF progenitors are unipotent and give rise to either cardiomyocytes or endothelial cells,
whereas SHF progenitors are either unipotent or bipotent consistent with a temporal differentiation delay between FHF and
SHF during the progressive restriction of fate potential by the
cardiogenic mesoderm progenitors.48 Therefore, the endocardium is essentially specified at gastrulation in mice although
some of the endocardium in the OFT and RV may originate
from multipotent SHF precursors not yet committed at the cardiac crescent stage. In summary, the endocardium is a specialized endothelial subpopulation originated by vasculogenesis
from progenitor cells in the cardiac crescent. One important
issue is that the endocardium expresses specific markers that
are not expressed in endothelial cells. For example, the transcription factor, NFATc1, expressed in the nucleus of endocardial cells since the beginning of endocardial differentiation49
is not required for early endocardial specification but is necessary for later valve morphogenesis.49,50
Endocardial Patterning
Notch patterns the early endocardium, which expresses several Notch elements and whose interaction with the myocardium is critical for cardiac development. Initial functional
analysis was performed on mice with systemic Notch signaling inactivation (RBPJk or Notch1 targeted mutants) in which
the expression of early valve and chamber endocardial markers was disrupted, suggesting impaired endocardial differentiation at a time when myocardial patterning was unaffected.3,24
Gain-of-function (GOF) experiments, in contrast, show that
ectopic N1ICD expression throughout the endocardium extends the uniform N1ICD distribution observed in prospective
valve tissue, thus expanding valve patterning to ventricular
endocardium.51
A key feature of Notch signaling in the midgestation
mouse heart (E9.5–10.5) is that endocardial cells express
the ubiquitin ligase Mib14 and both the Dll4 ligand and the
Notch1 receptor3,24,37 and thus have the potential to send and
receive signals. This is true for the presumptive valve territory
(AVC and OFT) and for the early ventricle. Notch inactivation
in the endocardium results in decreased ligand expression.
Thus, RBPJk and Notch1 mutants show reduced Dll4 expression in the endocardium,3,24 whereas Notch GOF expands Dll4
expression throughout this tissue,51–53 suggesting the existence
of a Notch-dependent positive feedback loop regulating Dll4
expression in the embryonic endocardium. These results imply that Notch-active endocardial cells behave as a developmental field and are specified together as valve or chamber
endocardium. This situation contrasts with findings in the
central nervous system54 and other tissues.55 In the following
sections, we will describe how endocardial Notch activity patterns the various cardiac territories.
Early Valve Development
Early valve development begins with the formation of the
primitive valve primordium by a process of regionalized EMT
that takes place initially in the AVC and later in the OFT endocardium to produce cushion mesenchyme (Figure 4A and
4B). Notch pathway genes are expressed in prospective valve
endocardium.24,37 Targeted inactivation of Notch1 (or RBPJk)
results in severely hypoplastic ECs at E9.5 because of impaired EMT24 (Figure 4C). Transmission electron microscopy
analysis revealed that although E9.5 RBPJk-deficient AVC
cells show features of activated premigratory endocardium,
they remain in close association, maintain adherens junctions,
and fail to detach from each other and migrate individually
to invade the cardiac jelly.24 The proposed mechanism is that
N1ICD-RBPJK directly activates the EMT drivers Snail1/2,
whose expression is severely reduced in RBPJk mutant AVC
and OFT endocardium.24,25 Loss of Snail expression prevents
downregulation of vascular endothelial cadherin (Cdh5)–
mediated endocardial cell adhesion and EMT is blocked
(Figure 4D). These in vivo observations were confirmed by
the defective EMT of AVC and OFT explants from Notch1
or RBPJk mutants or wild-type (WT) explants treated with a
Notch signaling inhibitor, cultured in vitro on collagen gels,
indicating that Notch is essential for this process.24 Functional
analysis has revealed the requirement of the transcription
factor Hey256 or the synergistic effect of Hey1 and Hey2
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Figure 4. Developmental processes in cardiac valve development. A, Diagram of an E9.5 heart. B, Detail of the boxed area in
A highlighting the outflow tract (OFT) and atrioventricular canal (AVC) endocardial cushions where epithelial–mesenchymal transition
(EMT) takes place and valves form. Myocardium, brown; endocardium, red; extracellular matrix, gray. During EMT, the AVC cushion tissue
is invaded by endocardial-derived mesenchyme (light blue), whereas the OFT is invaded by migratory neural crest (NC) mesenchyme
(purple) and endocardial-derived mesenchyme (light blue). The interaction between NC-derived and endocardial mesenchyme is
crucial for OFT valve morphogenesis. C, Cross section of a WT E9.5 heart (left) showing the 4 developing chambers. The arrow
points to a forming trabecula and the arrowhead to mesenchyme cells in the AVC; in the Notch1 mutant heart (middle), trabeculae are
poorly developed (arrow) and the AVC is devoid of mesenchyme (asterisks). Ectopic N1ICD expression in the endocardium of E9.5
R26N1ICD;Tie2-Cre embryos (right) causes impaired trabeculation (arrow), and mesenchyme cells distribute along a large AVC area.
D, Signaling circuitry regulating formation of the valve primordia via EMT. Cartoon depicting the E9.5 OFT and AVC. Myocardium, brown;
endocardium, red. AVC myocardial Bmp2 is required for Tgfβ2, Notch1, Snail1, Snail2, and Twist1 expression. Mib1-Dll4(Jag1)-Notch1
signaling occurring in a field of OFT or AVC endocardial cells leads to Snail1/2 activation and Bmp2 repression in endocardium via Hey
proteins. Snail1/2 in turn repress Cdh5 so that EMT occurs. Endocardial Notch activity leads to Wnt4 expression that in turn promotes
myocardial Bmp2 expression and EMT. E, Pathway establishing chamber vs valve domains in the developing heart. Myocardial Hey1
and Hey2 (green) confine Bmp2 expression to prospective valve myocardium (purple). Bmp2/Smad signaling together with Gata4 drive
valve-specific gene expression. Endocardial Notch signaling (red) activates Hey1–HeyL, which repress Bmp2 in the endocardium. F, Top,
Semilunar (SL) valves morphogenesis. From E13.5 onward, once mesenchyme cells occupy the endocardial cushion, cusps (Continued )
e8 Circulation Research January 8, 2016
Figure 4 Continued. excavation is driven by a solid endothelial ingrowth of the free edges of the valve cusps on their arterial face,
producing an epithelial ridge between the emerging cusps and the arterial wall. The ridge eventually becomes luminated to yield the sinus
of Valsalva. Bottom, AV valves morphogenesis. The mural leaflets of both AVV are supported by AV myocardium at their ventricular side.
As the primordial leaflets distend into the lumen, thin strands of elongated muscle remain attached to the valve leaflet and programmed
cell death yields a mobile leaflet and remnants that contribute to the chordae tendinae and papillary muscles. Myocardium is depicted in
brown, proteoglycans in pink, and mesenchyme in gray. la indicates left atrium, lv, left ventricle; ra, right atrial; rv, right ventricle; and WT,
wild-type.
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abrogation57,58 or Hey1 and HeyL in valve development.59 The
endocardial expression of these genes responds to Notch signaling manipulation,24,51 suggesting that they are Notch targets
in this tissue (Figure 4D).
In complementary Notch1 GOF studies, constitutive
Notch1 activity in the endocardium enables ectopic, noninvasive EMT of ventricular endocardial cells, conferring valvular features to an otherwise nonvalvular, EMT-refractory
ventricular endocardium.51 Notch GOF studies also revealed
the integration of endocardial Notch and myocardial Bmp2
signaling60,61 to promote EMT: Notch is required for Snail
expression and Bmp2 signaling regulates Snail activation,
suggesting that both pathways converge to activate cardiac
EMT.51 Recent studies with conditional loss-of-function
(LOF) alleles have confirmed previous findings and have
added complexity to the model, suggesting that endocardial
Notch1 signaling induces the expression of Wnt4, which subsequently acts as a paracrine factor to upregulate Bmp2 expression in the adjacent AVC myocardium to signal EMT62
(Figure 4D). Jag1 has also been suggested to be required for
EMT as Jag1flox;VE-Cadherin-Cre,63 and Jag1flox;Nfatc1-Cre
mice62 show hypocellular EC in vivo and in vitro. Because the
majority of Jag1flox;VE-Cadherin-Cre mice survive to adulthood, the absolute requirement of Jag1 in valve primordium
formation is debated. Our current data (MacGrogan et al, in
preparation) suggest that Mib1 acting in the endocardium
ubiquitylates Dll4 (and perhaps Jag1) in signaling cells so that
productive ligand–receptor interaction occurs, and the Notch1
receptor is activated in neighboring endocardial cells leading
to downstream events leading to EMT both in AVC and OFT
(Figure 4D).
Notch and Hey transcription factors regulate Bmp2 expression to pattern valve versus chamber territories in the
heart. Thus, data from Notch GOF and LOF51,53 together with
Hey1 and Hey2 GOF and LOF52,64 studies indicate that Bmp2
expression is confined to the AVC myocardium by Hey1 and
Hey2 both of which expressed in the chambers.52,64 In the endocardium, Notch1-dependent Hey1, Hey2, and HeyL are differentially expressed in the AVC chambers and repress Bmp2.
Ectopic N1ICD expression in endocardium expands AVC patterning to ventricular endocardium but does not affect Hey1,
Hey2, or Bmp2 myocardial expression. Ectopic N1ICD expression in the myocardium leads to expansion of Hey1 and
loss of Bmp2 expression, and chambers’ markers expand to
the AVC.51 In both N1ICD GOF models, Bmp2 is repressed
in the endocardium, similar to the WT situation. Systemic
abrogation of Notch signaling leads to downregulation of
Hey genes and ectopic endocardial Bmp2 expression, indicating that Notch represses Bmp2 in the endocardium via Hey
proteins (Figure 4E).51 The complexity of the signaling and
transcription factor network regulating AVC-specific gene expression and patterning is suggested by recent work showing
that the Gata4 transcription factor together with Bmp2/Smad
signaling activates AVC-specific enhancers, leading to H3K27
acetylation and subsequent AVC-myocardium–specific gene
expression (Figure 4E).65 AVC patterning is also essential for
delay of electric impulses between atria and ventricles, and
defective AVC maturation can lead to arrhythmias such as
AV re-entry tachycardia, AV nodal block, and ventricular preexcitation. Wnt and Notch signaling converge to regulate AVC
maturation and electric programming upstream of Tbx3.66
OFT Development and Remodeling
The OFT at the arterial pole of the heart is a transient structure that connects the embryonic ventricles to the aortic sac.
The OFT is elongated by addition of SHF progenitors and subsequently as the circulatory system transits from a single to a
double pathway; the OFT rotates clockwise and the aorta and
pulmonary orifices reposition above the left and right ventricles,
respectively. These morphogenetic movements depend on the
coordinated interactions between multiple cell types originating from within and outside the heart: the endocardium, endocardial-derived mesenchyme, SHF-derived cardiac and smooth
muscle progenitors, and cardiac neural crest (CNC).
Deficient SHF contribution to the OFT leads to a spectrum of malformations such as double outlet right ventricle
and over-riding aorta, which reflect a continuum of incorrect
alignment defects.67 These defects closely resemble those
seen in humans with AGS, a disorder caused by JAG139,40 or
NOTCH268 mutations and characterized by right-sided OFT
defects and disorders in the skeletal, ocular, renal, and hepatic
organ systems.69 JAG1 LOF mutations or reduced copy number of JAG1 and NOTCH1 have also been identified in nonsyndromic tetralogy of Fallot, which has 4 major components:
valvular or subvalvular pulmonary stenosis, ventricular septal defect (VSD), over-riding aorta, and RV hypertrophy.70,71
Systemic Jag1 deletion in mice results in embryonic lethality at E10.5, whereas heterozygous Jag1 mice exhibit eye defects but none of the other characteristic AGS phenotypes.72
However, mice double heterozygous for the Jag1 null allele
and a Notch2 hypomorphic allele exhibit characteristic AGS
abnormalities including heart developmental defects such
as hypoplastic RV and over-riding aorta.73 Accordingly, homozygous systemic deletion of PS1 which encodes for the
γ-secretase complex member Presenilin 1,74 or combined
Hey1;HeyL59 or Hes1 deletion,75 all display characteristic right
OFT defects although none of these genes have been linked
to congenital heart disease (CHD) in humans to date (Table).
OFT morphogenesis is also dependent on a second
source of extra cardiac cells derived from the CNC and required for OFT septation, formation of the arterial valves
(also called semilunar [SLVs] because of the shape of their
leaflets, which are also called cusps) and remodeling of the
AAAs (Figure 2E). Deficient CNC investment into the OFT
Luxán et al Endocardial Notch Signaling e9
Table. Mutations in Notch Pathway Elements Affecting Heart Development in Humans and Mice
Mouse Model
Phenotype
Defective Endocard. Human Gene
Signaling
Disease
Mechanism of
Human Disease
References
Downloaded from http://circres.ahajournals.org/ by guest on May 4, 2017
Notch1+/−
Valve calcification
Yes
NOTCH1
CAVD, BAV, TOF
HI
41
Notch1−/−
Hypoplastic EC and impaired trabeculation
Homozygous lethal (E10.5)
Yes
…
n/a
…
3, 24
Notch1flox/flox;Tie2-Cre
Hypoplastic EC and impaired trabeculation
Homozygous lethal (E10.5)
Yes
…
n/a
…
3
Notch1flox/flox;Nfatc1pan- Hypoplastic EC
Cre
Homozygous lethal (E12.5)
Yes
…
n/a
…
62
Notch2del1/+
Hypomorphic allele, AGS phenotype in Jag1+/dDSL;
Notch2+/del1 double heterozygous
(see below)
n.d.
NOTCH2
AGS
HI
68, 73, 76, 77
Notch2del1/del1
Myocardial hypoplasia and reduced trabeculation
Homozygous lethal (E10.5)
n.d.
NOTCH2
n/a
…
68, 76, 77
Notch2flox/ex;Pax3-Cre;
Narrow aortas and pulmonary arteries
n.d.
…
AGS (PAS)
…
78
Notch2flox/ex;Tag1-Cre
Narrow aortas and pulmonary arteries
Neonatal lethality
n.d.
…
AGS (PAS)
…
78
RBPJk−/−
Defective cardiac looping, hypoplastic EC and
impaired trabeculation
Homozygous lethal (E10.5)
Yes
RBPJK
n/a
…
3, 24
RbpJk+/−
Aortic valve sclerosis, stenosis
Yes
…
n/a
…
79
Impaired trabeculation
Homozygous lethal (E10.5)
Yes
…
n/a
…
3
RPJkflox/flox;Dermo1-Cre
Ventricular septal defect
Yes
…
n/a
…
80
Dll4
Reduced atrial and ventricular chambers,
defective trabeculation
AV malformations Homozygous lethal (E10.5)
Yes
DLL4
n/a
…
81, 82
Jag1−/−
Pericardial edema
Homozygous lethal (E11.5)
n.d.
JAG1
AGS
(heterozygous
mutations)
HI
39, 40, 72
Jag1+/−;Notch2+/del1
RVHP, AVSD, VSD, PAS
Perinatal lethality
n.d.
JAG1;NOTCH2
AGS
HI
39, 40
Jag1flox/flox;Cdh5-Cre
TOF, valve calcification
Yes
…
n/a
…
63
Jag1flox/flox;Nfatc1-Cre
Hypoplastic EC
Homozygous lethal (E11.5)
Yes
…
n/a
…
62
Jag1flox/flox;Mef2c-AHFCre
AA abnormalities, VSD, ASD
Homozygous neonatal lethal
Yes
…
n/a
…
83
Jag1flox/flox;Islet1-Cre
AA abnormalities, VSD, ASD
Homozygous neonatal lethal
Yes
…
n/a
…
83
Mib1flox/flox;cTnT-cre
LVNC
Yes
MIB1
LVNC
HI
DN
4
Psen1−/−
VSD, DORV, PAS
Yes
PSEN1,
PSEN2
Dilated
cardiomyopathy
…
74, 84, 85
Psen1−/−;Psen2−/−
Defective cardiac looping, Homozygous lethal
(E10.5)
n.d.
PSEN1,
PSEN2
n/a
…
86
Hey2−/−
VSD, ASD, PAS, TOF, TVA, AVV dysfunction
Yes
HEY2
n/a
…
56, 58, 87, 88
Hey1 ;Hey2
Defective cardiac looping, hypoplastic EC and
impaired trabeculation, homozygous postnatal
lethality
Yes
HEY1, HEY2
n/a
…
57, 89
Hey1−/−;HeyL−/−
VSD, AVV defects
Homozygous perinatal lethality
Yes
HEY1, HEY3
n/a
…
59
Hes1−/−
VSD and OA
Homozygous lethal (E18.5)
Yes
HES1
n/a
…
75, 90
DNMAML;Pax3-Cre
OFT, AA abnormalities, VSD, postnatal lethality
…
MAML
n/a
…
91
DNMAML;Wnt1-Cre
OFT, AA abnormalities,
postnatal lethality
…
…
n/a
…
91
RBPJk
;Tie2-Cre
flox/flox
−/−
−/−
−/−
(Continued )
e10 Circulation Research January 8, 2016
Table. Continued
Mouse Model
Phenotype
Defective Endocard. Human Gene
Signaling
Disease
Mechanism of
Human Disease
References
Downloaded from http://circres.ahajournals.org/ by guest on May 4, 2017
DNMAML;Islet1-Cre
OFT, AA abnormalities, VSD, TVA, neonatal
lethality
*
…
n/a
…
83
DNMAML;Mef2c-AHFCre
OFT, AA abnormalities, VSD, neonatal lethality
*
…
n/a
…
83
Numbflox/flox;Numbl flox/flox; OFT septation defect, DORV, AVSD, embryonic
Nkx2.5-Cre
lethal (E15.5–18.5)
*
NUMB,
NUMBL
n/a
…
92
N1ICD ME;Mesp1-Cre
(NGOF)
Impaired ventricular myocardial differentiation,
ectopic trabeculation in AVC
Homozygous lethal (E10.5)
*
…
n/a
…
53
R26N1ICD;Tie2-Cre
(NGOF)
Expansion of valve features to ventricles,
ectopic partial EMT in ventricles
Homozygous lethal (E10.5)
Yes
…
n/a
…
51
R26N1ICD;Nkx2.5-Cre
(NGOF)
Hypoplastic EC and thin chamber myocardium,
defective trabeculation embryonic lethal (E11.0)
Yes
…
n/a
…
51
R26N1ICD;cTnT-Cre
(NGOF)
Hypoplastic EC and delayed trabeculation,
embryonic lethal (E11.5)
…
…
n/a
…
51
Hey1 ME;Mesp1-Cre
(HGOF)
Reduced AVC extension, embryonic lethal (E11.5)
Yes
…
n/a
…
64
Hey2 ME;Mesp1-Cre
(HGOF)
Absent AVC, no EC, defective trabeculation,
embryonic lethal (E10)
Yes
…
n/a
…
64
AA indicates aortic arch; AGS, Alagille syndrome; ASD, atrial septum defect; AVSD, atrioventricular septum defect; AVV, atrioventricular valve; BAV, bicuspid aortic
valve; CAVD, calcific aortic valve disease; DN, dominant negative; DORV, double outlet right ventricle; EMT, epithelial–mesenchymal transition; EC, endocardial cushion;
HI, haploinsufficiency; HGOF, Hey gain-of-function; LVNC, left ventricular noncompaction; MAML, Mastermind-like; Mib1, mind bomb-1; n.a., not-applicable; NECD, Notch
extracellular domain; NGOF, Notch gain-of-function; OA, over-riding aorta; PAS, pulmonary artery stenosis; RVHP, right ventricular hypoplasia; TOF, tetralogy of Fallot; TVA,
tricuspid valve atresia; and VSD, ventricular septal defect.
*Hypothetical.
results in persistent truncus arteriosus because of lack of mesenchymal contribution to the aortic-pulmonary septum and
a perimembranous VSD, which results from the inappropriate growth and fusion of the conotruncal ridges of the outlet
septum and ECs of the inlet septum.67 Moreover, CNCs play
essential roles in positioning the cushions and patterning the
valve cusps within the developing outflow cushions.93 CNCs
affect the deployment of SHF progenitors and are involved
in cross talk with other pharyngeal mesoderm progenitors,
including endothelial cells, as they approach the distal heart
tube. Normally CNCs come in close contact with SHF cells as
they migrate into the OFT, and positional cues are exchanged
by cell–cell interactions. Conditional inhibition of Notch signaling using a dominant-negative form of the transcriptional
coactivator mastermind (Figure 1) induced with the CNC
driver lines Wnt1-Cre or Pax3-Cre resulted in a variety of defects that appear with variable penetrance: pulmonary artery
stenosis, VSD, atrial septum defect, and OFT misalignment
similar to tetralogy of Fallot.91 Analogous to the SHF situation, the number, specification, or migration of CNCs was
not altered by Notch ablation, but differentiation into smooth
muscle cells surrounding the AAAs was greatly diminished.91
Likewise, deletion of the Jag1 ligand or Notch signaling inhibition by dominant-negative mastermind-like using either
Isl1-Cre or Mef2c-AHF-Cre driver lines resulted in double
outlet right ventricle persistent truncus arteriosus, VSD, and
AAA typical of the CNC.83 The development of neighboring
tissues was affected including abnormal migration of CNCs
and defective EMT, suggesting that Notch is an important
mediator of interactions between SHF, CNC, and OFT endocardium/endothelium.83 In this regard, Fgf8 is a key molecule
required for coordinating CNC ingression and endocardial
EMT during OFT remodeling, and Fgf8 may act upstream of
Bmp4 in these processes (Figure 4D).94 In support of this notion, the EMT defect seen in Isl1-Cre;DNMAML mice could
be rescued by supplying recombinant Fgf8 in explant assays,
indicating that functionally the effects of Notch signaling in
the SHF are mediated at least, in part, by Fgf8.83
Deleting Jag1 in the endothelium using a VE-cadherin-Cre
line that drives recombination with variable efficacy but with
minimal activity in the hematopoietic cell lineage recapitulated many of the phenotypes typically associated with Notch
SHF or CNC LOF mutants.63 The mutant mice resultant from
endothelial Jag1 deletion displayed cardiac defects that were
highly reminiscent of those present in AGS, including RV hypertrophy, VSD, over-riding aorta, valve malformations, and
stenosis. Absence of endothelial Jag1 caused partial blockage
of EMT and myxomatous valves, bone formation, and valve
calcifications between 5 and 13 months of age.63 Thus, defects
manifested in the Notch SHF or CNC LOF mutants and in
AGS patients may be ascribed, in part, to defective signaling
in the endocardium/endothelium.
Valve Morphogenesis and Aortic Valve Disease
By the time it reaches its definitive length, the OFT is formed
by mesenchyme derived from the endocardium proximally
Luxán et al Endocardial Notch Signaling e11
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(conus) and CNCs distally (truncus).95 Concurrent with rotation and septation of the OFT, the aortic and pulmonary valves
form at either side of the conotruncal boundary by progressive
enlargement and fusion of the EC.96,97 The outflow cushions
excavate gradually from E12.5 to E15.5, to achieve their typical
semilunar morphology (Figure 4F).98 On the inflow side, fusion
of ECs at the AV junctions yields a right and left AVC. Through
a variety of mechanisms (excavation or delamination), the
fused cushions eventually yield the leaflets and cords of the
tension apparatus.95,99 Thus, the chordae tendinae and the fibrous continuity separating the mitral and tricuspid valves in
addition to the leaflets arise from EC endothelial cells.100,101
These valvulogenic processes are driven by a combination
of endocardial gene expression programs and biomechanical forces elicited by evolving hemodynamics, but the precise
mechanisms are unclear.102
Although the role of Notch signaling during EMT is well
documented, Notch function in post-EMT morphogenesis
remains uncertain. Nevertheless, in light of the association
with BAV and calcification, there has been significant interest
in elucidating the potential role of Notch1 in EC fusion and
remodeling. Strikingly, the expression of N1ICD in ECs and
remodeling valve endocardium throughout late developmental stages suggests that the endocardium is the primary site
for Notch activity in the ECs post EMT.37 Given these considerations a relevant question is what are the mechanism(s)
involved in the transition from proliferation and expansion of
the ECs to remodeling and elongation of the valves cusps and
leaflets? Throughout valve development, endocardial Notch is
likely required to intersect with other pathways to coordinate
valvulogenesis.6,103 Several developmental signaling pathways
have been identified in the cushions to regulate valve remodeling including members of the Bmp and Egfr pathways.104
For example, global or endothelial deletion of the heparinbinding epidermal growth factor results in normal development, but excessive enlargement of the conal cushions caused
by increased proliferation and prolonged Smad1/5/8 phosphorylation in cushion mesenchyme.105,106 Accordingly, this
defect is phenocopied in BMP GOF mouse models lacking
the Bmp-specific nuclear inhibitor Smad6107 or the BMP antagonist Noggin.108 Recent evidence from our group indicated
that endocardial-specific disruption of Mib1, Jag1, or Notch1
results in dysplastic and bicuspid valve accompanied by increased Bmp signaling and proliferation resulting in thickened
SLV cusps and AV leaflets (MacGrogan et al, in preparation).
We found that Jag1 can transcriptionally upregulate heparinbinding epidermal growth factor, which released in a soluble
form associates with the valve extracellular matrix and inhibits
mesenchymal cell proliferation through epidermal growth factor receptor.109 This suggests that endocardial Notch signaling
functions during valve morphogenesis to regulate Egfr and
Bmp signaling and thus limit valve cushion proliferation before remodeling. Importantly, SLV cusps remodeling may also
depend on appropriate tissue–tissue interactions among the
SHF and CNC. In addition to the previously described OFT
defects, inhibition of Notch in the SHF using the dominantnegative mastermind-like transgene resulted in dysmorphic
and thickened SLV cusps and aortic regurgitation caused by
diminished mesenchymal apoptosis and increased extracellular matrix deposition.110 Given that cardiovascular defects in
the Notch SHF mutants occur, in part, because of abnormal
CNC patterning, CNCs were interpreted to provide instructive
signals to orchestrate apoptosis and changes in extracellular
matrix production during SLV remodeling.110
SLV malformations are common in humans, including
BAV, which occurs in 0.5% to 3% of the population, and pulmonic valve stenosis in 10% of all CHD in adults.111 BAV is
usually asymptomatic at birth but increases the risk of disease
later in life, including calcification, stenosis, and aortopathy.112
BAV morphology varies between right and left (R–LC) coronary cusp fusion and noncoronary (R–NC) fusion at a ratio of
≈0.7 to 0.3 (Figure 2H). Insight into the developmental basis
of BAV was gained from studies of Nos3 null mice and an inbred Syrian hamster strain, suggesting that R–N BAV is caused
by defective formation of noncoronary cushions, whereas the
R–L BAV is likely the result of defective OFT septation.113 To
date, only NOTCH1 has been implicated in the cause of nonsyndromic BAV in human. In a study of 2 unrelated families,
Garg et al41 observed mutations in NOTCH1 that segregated
with aortic valve disease, particularly with BAV and aortic
valve calcification. In addition to BAV, other forms of LV OFT
obstruction such as coarctation of the aorta and hypoplastic
left heart have been related to NOTCH1 polymorphisms and
mutations in the extracellular domain of NOTCH1.41,114 The
mechanism of calcific aortic valve disease was proposed to be
haploinsufficiency of NOTCH1 resulting from failure to repress a pro-osteogenic gene program in the valves mediated by
Runx2 upregulation through the Hey genes.41 Recent in vitro
modeling of calcific aortic valve disease in endothelial cells
derived from human-induced pluripotent stem cells extended
these findings to multiple epigenetic and transcriptomic targets
affected by NOTCH1 haploinsufficiency.115 Thus, hemodynamic shear stress, which protects against calcification, activates
antiosteogenic gene networks and represses proinflammatory
molecules in WT but not in NOTCH1 mutant endothelial cells,
consistent with the notion that the effects of shear stress are
mediated, in part, by NOTCH1. This study further showed that
heterozygous nonsense mutations in NOTCH1 disrupted several epigenetic marks, including H3K27ac at N1ICD-bound
enhancers resulting in derepression of latent anti-inflammatory
and pro-osteogenic gene networks.115
Ventricular Trabeculation
Ventricular chamber development begins in the mouse around
E9.5. The morphological landmark of this process is the formation of trabeculae, a mesh of endocardium-lined luminal
cardiomyocyte projections that give the embryonic myocardial walls their characteristic spongy appearance.31,116
Trabecular cardiomyocytes and endocardium are in close apposition, especially at the base of the forming trabeculae, and
communication between both tissue layers regulates oriented
trabecular growth and patterning.117–120 Trabeculae increase
cardiac output and permit nutrition and oxygen uptake in the
embryonic myocardium before coronary vascularization116,120
and failure in trabeculae formation is embryonic lethal.121–123
Cells at the tip of the longitudinal axis of the trabeculae are
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more differentiated than those at the base.124 As development
proceeds, trabeculae extend radially and form a network
while also increasing in length.116 Progressively, the ventricular myocardium differentiates into 2 distinct layers: the outer,
proliferative, compact myocardium and the inner, more differentiated, trabecular myocardium.124
Systemic or endocardium-specific Notch mutant embryos
show impaired trabeculation, defective trabecular marker expression and reduced ventricular cardiomyocyte proliferation,
leading to the formation of an anomalous spongy myocardial
wall (Figure 5A and 5B). These findings suggest that endocardial Notch1 signaling is required for proliferation and differentiation of chamber myocardium.3,125,126 Notch signaling
abrogation affects the expression of 3 signaling pathways required for trabeculation: Bmp10, Nrg1/ErbB, and EphrinB2.
Bmp10 is expressed in trabecular myocardium where it sustains proliferation.121 Phenotypic rescue of RBPJk mutant
embryos after incubation in Bmp10-conditioned medium
suggests that in trabecular myocardium, Notch modulates
proliferation via Bmp10.3 Endocardial Nrg1 is crucial for trabeculation122 via activation of ErbB2-4 receptors expressed on
myocardium.127 Addition of Nrg1 to cultured RBPJk mutant
embryos rescues their cardiomyocyte differentiation defect.3
The EfnB2 ligand and EphB4 receptor signaling system is
also required for trabeculation.128 Endocardial EfnB2/EphB4
expression and activity is impaired in Notch mutants, and
EfnB2 is a direct transcriptional target of N1ICD/RBPJK.3
Studies with targeted mutant embryos indicated that Nrg1
transcription is reduced in EfnB2 mutants (but not vice versa), whereas Bmp10 mutants show normal EfnB2 and Nrg1
expression, suggesting that EfnB2 acts upstream of Nrg1 and
that both molecules may act independently of Bmp10 during
trabeculation. Recently, the Hand2 transcription factor has
been shown to be an important downstream component in the
Notch-dependent endocardium–myocardium signaling mechanism active in trabeculation, and a direct regulator of Nrg1.129
Additional work has shown that during trabeculation the endocardial activity of the peptidyl-prolyl isomerase FKBP12
tightly regulates N1ICD stability.130 Our current data suggest
a model in which Mib1-Dll4-Notch1 signaling in the endocardium mediates an endocardium–myocardium interaction
that promotes cardiomyocyte proliferation and differentiation
leading to trabeculae formation (Figure 5C).
LV Noncompaction
Formation of the trabecular network is followed by a period
of remodeling at E14.5 called compaction, when trabeculae
stop growing and thicken radially until they are no longer
distinguishable from the myocardial wall. Simultaneously,
epicardium-derived cells131 invade the compact myocardium
and, together with ventricular endocardial cells,5 contribute
to the formation of the coronary vasculature that will support
the rapid proliferation of the compact myocardium, which
becomes the main source of contractile force of the heart.132
Several pathways are crucial for these developmental processes, in which signals derived from endocardium and myocardium interplay with others derived from coronary blood
vessels to coordinate ventricular myocardium patterning,
proliferation, trabecular maturation/compaction, and coronary
vessel formation.120
Defective trabecular compaction can manifest as a cardiomyopathy termed LVNC (OMIM 601493), characterized by
the presence of prominent trabeculations separated by deep
invaginations in an abnormally thin ventricular wall.133,134
LVNC can be a mild disease revealed by a depressed systolic
function135 or can evolve into a serious condition with complications including systemic embolism, malignant arrhythmias, heart failure, and sudden death.136 LVNC is generally
transmitted as an autosomal dominant trait, but the underlying
molecular mechanisms are still poorly understood.134
Conditional inactivation in the embryonic mouse myocardium of the Mib1 gene, encoding the E3 ubiquitin ligase
Mib1 essential for Notch ligand signaling14 (Figure 1), causes
a phenotype strongly reminiscent of LVNC. Thus, E16.5
Mib1flox;cTnT-Cre embryos show a dilated heart with a thin
compact myocardium and large, noncompacted trabeculae,
protruding toward the ventricular lumen4 (Figure 5D and 5E).
These embryonic features are present in newborn and adult
mutant mice as revealed by echocardiographic analysis of
6-month-old mice that also shows reduced ejection fraction
and a high ratio of noncompacted to compacted myocardium,
diagnostic of LVNC in humans.137
Sequencing of the MIB1 gene in a cohort of 100 LVNC
patients (48% familial cases) identified 2 mutations (V943F
and R530X) in 2 probands. The 2 MIB1 mutations were inherited in an autosomal dominant fashion across various generations, together with LVNC. Image analysis of the hearts
of both probands’ fathers as well as those of family members
with inherited MIB1 mutations demonstrates the presence of
prominent trabeculations in both ventricles and reduced cardiac performance. Importantly, analysis of peripheral blood
from these patients reveals significantly below-normal expression of NOTCH1 and its targets DTX1 and GATA3. These data
demonstrate that the 2 MIB1 mutations affect NOTCH signaling and segregate with LVNC.4
In silico modeling suggests that MIB1 functions as a homodimer, in which MIB1 monomers form a head-to-tail homodimer that interact with JAG1 through the Herc2 domain.4
Moreover, FRET signal measurements and coimmunoprecipitation studies showed that WT MIB1 monomers dimerize and
that dimers are also formed between WT and mutant MIB1
monomers or between mutant monomers. Both mutations
result in loss of MIB1 WT function. In the case of R530X,
this would be because of haploinsufficiency caused by insufficient synthesis of WT MIB1 protein; in the case of V943F,
this would be because of a dominant-negative effect of the
mutant protein, titrating down the amount of functional WT
MIB1 dimers through heterotypic or homotypic interactions.
In both cases, loss of MIB1 function leads to disease inherited
in an autosomal dominant fashion.4
Analysis of chamber-specific markers in Mib1flox;cTnT-cre
mutant embryos revealed the expansion of compact myocardium markers to the trabeculae and reduced expression of trabecular markers. Global gene expression analysis confirmed
these results and also showed that genes involved in the differentiation of endocardium, trabecular cardiomyocytes, and
Luxán et al Endocardial Notch Signaling e13
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Figure 5. Endocardial Notch signaling is essential for chamber development. A, Transverse section of an hematoxylin and eosin–
stained WT E9.5 heart at the level the left ventricle (lv). The arrow points to a forming trabecula. B, The heart of a E9.5 RBPJk mutant
embryo shows a collapsed endocardium (arrowhead) and poorly developed trabeculae (arrow). C, Scheme depicting Notch pathway
elements expression the early ventricular myocardium: Mib1 is expressed throughout the endocardium (purple), Dll4 in endocardium at
the base of the forming trabeculae (green), where Notch1 is activated (red), triggering signals involved in cardiomyocyte proliferation and
differentiation. At this stage, the primitive myocardial epithelium begins to express compact myocardium markers. Images derived from
Grego-Bessa et al.3 D, Episcopic 3-dimensional reconstruction of E16.5 WT and E, Mib1flox;cTnT-Cre hearts. Note the thick ventricular
walls (brackets) and the small trabecular ridges (arrows) in the WT heart (D) and the thin compact myocardium and noncompacted
trabecular in the mutant heart (E). *Disorganized interventricular septum. F, Schematic representation of Notch function during ventricular
maturation and compaction. Mib1-Jag1 signaling from the myocardium (blue) activates Notch1 throughout the endocardium (red) to
sustain trabecular patterning, maturation, and compaction. Proliferative compact myocardium is defined by the expression of Hey2,
Tbx20, and n-myc, whereas the nonproliferative trabecular myocardium is defined by the expression of Anf, Bmp10, and Cx40. Images
derived from Luxán et al.4 la indicates left atrium, ra, right atrial; and rv, right ventricle.
coronary vessels were downregulated in mutant embryos.
In this regard, Bmp10 was downregulated in Mib1flox;cTnTcre mutants, similar to the situation in endothelial-specific
RBPJk mutants,3 indicating that endocardial–myocardial
Notch signaling is crucial first for trabeculae formation and
later for trabecular maturation and compaction. The observation that compact zone markers are expanded to the trabeculae of E15.5 Mib1flox;cTnT-cre mutant mice further suggests
that trabecular patterning and maturation are impaired. Our
working model suggests that myocardial Mib1 activity
e14 Circulation Research January 8, 2016
enables Jag1-dependent activation of Notch1 in the endocardium, triggering downstream signaling events that sustain
normal trabecular patterning, maturation, and compaction
over a developmentally regulated time window (Figure 5F).
Abrogation of Mib1-dependent signaling in the myocardium
disrupts chamber maturation and compaction, resulting in
LVNC. These data establish the causal role of NOTCH deregulation in LVNC.
Endocardial Notch Signaling in Zebrafish
Heart Regeneration
Downloaded from http://circres.ahajournals.org/ by guest on May 4, 2017
The adult zebrafish heart exhibits a remarkable capacity to
regenerate after ventricular resection138 or cryoinjury-induced
myocardial infarction.139–141 The cryoinjury model triggers
similar processes to those occurring in the mammalian heart
after an ischemic injury: cardiac cell death, inflammatory cell
infiltration, and the deposition of a fibrin and collagen-rich
scar. The fish heart dissolves this scar, and injured tissue is
replaced by new cardiomyocytes generated from dedifferentiation139–141 as seen in the resection model.142,143
Examination of Notch expression in the zebrafish heart
after cryoinjury shows a rapid upregulation of Notch signaling in the endocardium at the injury site (J. Münch, PhD, et al,
unpublished observations, 2015), in agreement with reports
of Notch reactivation after cardiac resection.144,145 Functional
and gene expression analysis in the regenerating heart reveals
that endocardial Notch activity is required for not only cardiomyocyte proliferation as reported in the ventricular resection
model144 but also fibrotic tissue deposition and attenuation of
the inflammatory response. As regeneration proceeds, the endocardium forms a scaffold that facilitates the entry of newly
generated cardiomyocytes into the fibrotic area. Our work underscores the function of endocardial Notch signaling in cardiac regeneration as an important mediator between fibrotic
tissue deposition and cardiomyocyte proliferation at the inner
injury border (Münch et al, submitted).
Disease Modeling
Mutations in NOTCH signaling components in humans are
implicated in CHD. The generation and analysis through the
years of a great variety of targeted mutant mice harboring
standard and tissue-specific mutations in several Notch pathway components suggest that a large number of these genes
may be involved in CHD. This research also illustrates the
connection between the fields of developmental biology and
pathology. In the simplest situation (Mendelian inheritance)
and to understand disease mechanisms, one would wish to
find a direct correlation between the alterations caused in
humans by mutations in a given gene and the phenotypes of
targeted mutant mice for that same gene. This has not been always the case; the reasons for which could be multiple including the existence of as yet unidentified additional conditions/
factors involved in the disease (comorbidity), that the disease
is genetically complex, that the expressivity of the disease
phenotype is influenced by the epigenome, or that the use of
inbred strains in experimental studies causes a severe phenotype that is not paralleled in the human situation (or vice versa). For NOTCH, the correspondence between the human and
the mouse cardiac disease phenotypes has been satisfactory
to date. One illuminating example has been research in AGS,
in which the generation of Jag1 targeted mutant mice was initially somewhat disappointing because heterozygous animals
failed to exhibit the majority of the phenotypes associated
with AGS in humans.72 However, mice doubly heterozygous
for the Jag1 null allele and a Notch2 hypomorphic allele reproduced most of the clinically relevant phenotypes observed
in patients with AGS (Table).73,76 This finding led to the identification of NOTCH2 mutations in a cohort of JAG1 mutationnegative patients with AGS.68,77 Thus, animal model studies
shed light into how Notch2 and Jag1 mutations interact to create a more representative mouse model of AGS and provided
an explanation of the variable phenotypic expression observed
in patients with AGS.73
The advent of next-generation sequencing technology146
coupled with efficient DNA capture147 has enabled the use
of exome sequencing as a new approach to study the genetic
basis of human disease that combined with genome-wide association studies,148 is a powerful tool to identify the basis of
complex genetic traits. Genomic editing technologies such as
Crispr/Cas9 opens the door for the relatively rapid generation of inactivating mutations in new disease-candidate genes
identified by genome-wide association studies149,150 and the
generation of potentially pathogenic mutations identified by
exome sequencing150 or by any other next-generation sequencing–related approach.
Conclusions
In this review, we have described studies that uncover the role
of Notch during cardiac patterning, OFT morphogenesis, and
valve and chamber development and disease. These findings
are of not only fundamental biological interest but also have
important implications for understanding the cause of CHD.
Data from several laboratories indicate that (1) the endocardium is a crucial source of signals that play key roles in
cardiac development and disease; (2) the Notch pathway is a
paradigmatic endocardial-derived signal, which regulates cell
fate specification and tissue patterning in the early vertebrate
heart to define chamber versus valve domains; (3) endocardial
Notch and myocardial Bmp2 converge on Snail to activate
EMT, leading to valve primordium formation; (4) endocardial
Notch activity modulates in a non–cell autonomous manner
various myocardial signals (Tgfβ2, Bmp10) required for valve
and trabecular development; (5) during OFT and valve morphogenesis Notch is required for myocardial Fgf8 and Bmp4
expression and EMT and mediates the communication between endocardium- and NC-derived mesenchyme that results
in the mature valve; (6) Notch influences cellular proliferation, differentiation, and apoptosis during these processes; (7)
NOTCH1 mutations cause BAV leading to precocious calcific
aortic valve disease; (8) endocardial/endothelial NOTCH1 is
required for the activation of antiosteogenic molecules and repression of proinflammatory gene signatures in the adult valve;
(9) endocardial Notch is required for ventricular chamber development: during trabeculation, Mib1-Dll4-Notch1 signaling
regulates cardiomyocyte proliferation and differentiation, and
later Mib1-Jag1-Notch1 signaling promotes trabecular patterning, maturation, and compaction; (10) MIB1 mutations
Luxán et al Endocardial Notch Signaling e15
cause familial LVNC inherited in an autosomal dominant
fashion; (11) in the zebrafish regenerating heart, endocardial
Notch signaling mediates fibrotic tissue deposition and cardiomyocyte proliferation at the inner injury site. An important
direction for future work will be to uncover the mechanisms of
activation of the different tissue-specific Notch elements, both
in space and time, and to broaden the studies relating NOTCH
signaling alterations to clinical phenotypes.
Acknowledgments
We thank present and past members of the laboratory for their contribution to this review and apologize for the omission of studies not
discussed or cited because of space limitations. K. McCreath helped
in English editing.
Sources of Funding
Downloaded from http://circres.ahajournals.org/ by guest on May 4, 2017
J.L. de la Pompa is funded by grants SAF2013-45543-R,
RD12/0042/0005 Red de Investigación Cardiovascular (RIC) and
RD12/0019/0003 Red de Terapia Celular (Tercel) from the Spanish
Ministry of Economy and Competitiveness (MINECO) and a grant
from the BBVA Foundation for Research in Biomedicine. The Centro
Nacional de Investigaciones Cardiovasculares was supported by the
MINECO and the Pro-CNIC Foundation.
Disclosures
None.
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Endocardial Notch Signaling in Cardiac Development and Disease
Guillermo Luxán, Gaetano D'Amato, Donal MacGrogan and José Luis de la Pompa
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Circ Res. 2016;118:e1-e18; originally published online December 3, 2015;
doi: 10.1161/CIRCRESAHA.115.305350
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