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DOI: 10.1161/CIRCRESAHA.115.305350 1

REVIEW

Endocardial Notch Signaling in Cardiac Development and Disease

Guillermo Luxán1,2, Gaetano D'Amato1, Donal MacGrogan1, José Luis de la Pompa1

1Intercellular Signaling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovascular (CNIC), Melchor Fernández Almagro 3, E-28029 Madrid, Spain, and; 2

Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, D-48149 Münster, Germany.

G.L., G.D., and D.M. contributed equally to this manuscript.

Running title: Endocardial Notch Signaling

Subject Terms: Developmental Biology Myocardial Biology Animal Models of Human Disease Address correspondence to: Dr. José Luis de la Pompa

Intercellular Signaling in Cardiovascular Development and Disease Laboratory Centro Nacional de Investigaciones Cardiovascular (CNIC) Melchor Fernández Almagro 3 E-28029 Madrid Spain Phone: +34-620-936633 Fax: +34-91-4531304 [email protected]

by guest on July 9, 2018http://circres.ahajournals.org/

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DOI: 10.1161/CIRCRESAHA.115.305350 2

ABSTRACT

The Notch signaling pathway is an ancient and highly conserved signaling pathway that controls cell 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. Keywords: Notch, endocardium, cardiac valves, trabeculation, compaction, cardiomyopathy, cardiac development, signaling pathways, valve, ventricle, congenital cardiac defect. Nonstandard Abbreviations and Acronyms: AAA aortic arch arteries AGS Alagille syndrome ASD atrial septum defect AVC atrio-ventricular canal AVV atrio-ventricular valve BAV bicuspid aortic valve CAVD calcific aortic valve disease CC cardiac crescent CNC cardiac neural crest DNMAML dominant negative mastermind-like DORV double outlet right ventricle EC endocardial cushion ECM extracellular matrix EMT epithelial-mesenchymal transition FHF first heart field GOF gain-of-function HB-EGF heparin-binding EGF-like growth factor HI haploinsufficiency LOF loss-of-function LVNC left ventricular non-compaction NECD Notch extracellular domain NICD Notch intracellular domain OA overriding aorta OFT outflow tract PTA persistent truncus arteriosus SHF second heart field SLV semilunar valve TOF tetralogy of Fallot VSD ventricular septum defect WT wild type


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INTRODUCTION The assembly and function of a complex organ like 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 three 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 trabeculation 3 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-to-mesenchymal transition (EMT) to form the endocardial cushions (EC). The EC will form several important structures within the heart, including 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 crosstalk must take place early on to establish a tight functional coupling between the endocardium and 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. Then 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 later processes of OFT and valve morphogenesis and trabecular compaction. Finally, we provide examples of how Notch dysfunction in the endocardium results in cardiac structural malformations that can lead to disease in the neonate and adult, and speculate about the signaling and structural function played by the endocardium in the regenerating zebrafish heart.

The Notch Pathway.

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 (NECD) composed of a varied 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 convertase 10 (S1 cleavage, Figure 1), while sugars are added to the EGF-like repeats of NECD 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 non-covalent interactions. Once in the membrane, the NECD 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


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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 metalloproteases (Figure 1). 17, 18 The remaining Notch fragment becomes susceptible to cleavage by -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/RBPJK/Su(H)/Lag1; Figure 1).22 NICD–CSL binding displaces corepressors and histone deacetylases (HDAC) that repress target genes in the absence of Notch signaling, and allows recruitment of the transcriptional co-activator Mastermind-like (MAML). Formation of the CSL-NICD-MAML ternary complex allows recruitment of additional coactivators to activate transcription of target genes, typically those encoding repressor such as basic-helix-loop-helix (bHLH) transcription factors of the Hes and Hey families. 23 Nevertheless, the spectrum of direct Notch targets is very large and tissue-specific, including Snail1 24, 25, p21 26, c-Myc 27, EphrinB2 3 and Nrarp 28 (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 (CC, Figure 2A). 8 The crescent contains two 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 (ECM) termed cardiac jelly (Figure 2A,B). 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 morphogenesis 30 (Figure 2C,D) 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, while the SHF gives rise to the OFT myocardium and other parts of the heart except for the LV (Figure 2C,D). At E9.5, uneven growth within the heart tube results in ballooning of the chambers and establishment of the trabecular network 31 (Figure 2D). The endocardium lines the lumen of the cardiac chambers and through an EMT process forms the cardiac cushion mesenchyme in the atrio-ventricular 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 onwards the OFT is remodeled from a single OFT vessel which circulates blood into the three main aortic arch arteries to join two dorsal aortae that distribute the blood throughout the embryo. Neural crest emanating form the dorsal neural tube invade the aortic arch arteries to reach the OFT (Figure 2E) and differentiate into smooth muscle cells covering the aortic arch arteries endothelium (Figure 2E). Progressively, the aortic arch arteries 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 four-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 (AVV), and the cardiac fibroblasts and the endocardium gives rise to the cardiac valves and coronary vessel endothelium (Figure 2F,G). Defective endocardial Notch signaling affects cardiac valve and chamber development, leading to diseases such as bicuspid aortic valve formation (BAV) and left ventricular non-compaction cardiomyopathy (LVNC; Figure 2H,I). 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


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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 and throughout the myocardium (Figure 3A-A'',D and 38) while Dll4 is expressed in the endocardium (Figure 3B-B'',D and 3). Mib1 is expressed in both endocardium and myocardium 4 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-C'',D and 37). At E12.5, Jag1 is expressed in the myocardium around the semilunar valves and in the endocardium (Figure 3E,H) where Notch is active (Figure 3G,H). Dll4 expression in valve endocardium is greatly diminished compared to E9.5 (Figure 3F,H). In the chambers, myocardial Jag1 expression is maintained (Figure 3I,L), Dll4 expression is attenuated (Figure 3J,L), and Notch activity remains widespread in the endocardium (Figure 3K,L and 4). Once the general cardiac pattern is established, substantial cellular proliferation, tissue morphogenesis and terminal differentiation of the different cardiac structures occurs and the heart becomes fully functional at birth (Figure 2G). In late gestation, Jag1 is expressed in smooth muscle tissue around the valves (not shown) and in chamber myocardium (Figure 3M,P), Dll4 in developing coronary vessels (Figure 3N,P and 4) and Notch1 activity is persistently found in valve and chamber endocardium (Figure 3O,P). Defective NOTCH signaling has been shown to cause various cardiac pathologies including Alagille syndrome (AGS) 39, 40, BAV (Figure 2H and 41) and LVNC 4 (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.

The timing of endocardial specification and whether the endocardium is derived from progenitors that lack myocardial potential is controversial (reviewed in 42). Data from chick and zebrafish models involving retroviral single-cell tagging and tracking studies suggest that specification of endocardial and myocardial cells occurs prior to their ingression through the primitive streak during gastrulation, perhaps as early as the blastula stage and significantly prior to 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 CC stage.45 Moreover, while it was clear that the OFT endocardium and myocardium both derived from a common Flk1+ precursor distinct from endothelial cells 46 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 two 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 CC stage. In summary, the endocardium is a specialized endothelial subpopulation originated by vasculogenesis from progenitor cells in the CC. 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 differentiation 49 is not required for early endocardial specification but is necessary for later valve morphogenesis. 49, 50


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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 carried out 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 mid-gestation mouse heart (E9.5-10.5) is that endocardial

cells express the ubiquitin ligase Mib1 4 and both the Dll4 ligand and the Notch1 receptor 3, 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, while 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 CNS 54 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,B). 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 due to impaired EMT 24 (Figure 4C). Transmission electron microscopy analysis revealed that although E9.5 RBPJk-deficient AVC cells show features of activated pre-migratory 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 down-regulation 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 Hey2 56 or the synergistic effect of Hey1 and Hey2 abrogation 57, 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, non-invasive EMT of ventricular endocardial cells, conferring “valvular” features to an otherwise “non-valvular”, EMT-refractory ventricular endocardium. 51 Notch GOF studies also revealed the integration of endocardial Notch and myocardial Bmp2 signaling 60, 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


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expression in the adjacent AVC myocardium to signal EMT 62 (Figure 4D). Jag1 has also been suggested to be required for EMT as Jag1flox;VE-Cadherin-Cre 63 and Jag1flox;Nfatc1-Cre mice 62 show hypocellular EC in vivo and in vitro. As 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., MS 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 LOF 51, 53 together with Hey1 and Hey2 GOF and LOF 52, 64 studies, indicate that Bmp2 expression is confined to the AVC myocardium by Hey1 and Hey2 that are expressed in the chambers. 52, 64 In the endocardium, Notch1-dependent Hey1, Hey2 and HeyL are differentially expressed in the AVC and chambers and repress Bmp2. Ectopic N1ICD expression in endocardium expands AVC patterningtoventricularendocardium 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, similarly to the WT situation. Systemic abrogation of Notch signaling leads to down-regulation of Hey genes and ectopic endocardial Bmp2 expression, indicating that Notch represses Bmp2 in the endocardium via Hey proteins (Figure 4E and 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 electrical impulses between atria and ventricles, and defective AVC maturation can lead to arrhythmias such as AV re-entry tachycardia, AV nodal block, and ventricular pre-excitation. 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 (DORV) and overriding aorta (OA) which reflect a continuum of incorrect alignment defects. 67 These defects closely resemble those seen in humans with AGS, a disorder caused by JAG1 39, 40 or NOTCH2 68 mutations and characterized by right-sided OFT defects as well as 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 non-syndromic tetralogy of Fallot (TOF), which has four major components: valvular or subvalvular pulmonary stenosis, ventricular septal defect (VSD), OA and RV hypertrophy. 70, 71 Systemic Jag1 deletion in mice results in embryonic lethality at E10.5 while 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 OA. 73 Accordingly, homozygous systemic deletion of PS1 which encodes for the -secretase complex member Presenilin 1 74,


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or combined Hey1;HeyL 59 or Hes1 deletion 75, all display characteristic right OFT defects, although none of these genes have been linked to CHD in humans thus far (Table 1).

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 valves SLVs, because of the shape of their leaflets, which are also called cusps) and remodeling of the aortic arch arteries (AAAs; Figure 2E). Deficient CNC investment into the OFT results in persistent truncus arteriosus (PTA) due to lack of mesenchymal contribution to the aortic-pulmonary septum and a perimembranous VSD which results from the inappropriate growth and/or 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.76 CNCs affect the deployment of SHF progenitors and are involved in crosstalk 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 co-activator mastermind (DNMAML; 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, ASD and OFT misalignment similar to TOF.77 Analogous to the SHF situation, the number, specification or migration of CNCs was not altered by Notch ablation but the differentiation into SMC surrounding the AAAs was greatly diminished. 77 Likewise, deletion of the Jag1 ligand or Notch signaling inhibition by DNMAML using either Isl1-Cre or Mef2c-AHF-Cre driver lines resulted in DORV but also PTA, VSD and AAA typical of the CNC. 78 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. 78 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; reviewed in 79). 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. 78

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, OA, 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 lengththe OFT is formed by mesenchyme derived from the endocardium proximally (conus) and CNCs distally (truncus). 80 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. 81, 82 The outflow cushions excavate gradually from E15 through E15.5, to achieve their typical semilunar morphology (Figure 4F).83 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. 80, 84 Thus, the chordae tendinae and the fibrous continuity separating the mitral and tricuspid valves in addition to the leaflets, arise from EC endothelial cells. 85, 86These 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.87


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While 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 suggested that the endocardium was 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, 88 Several developmental signaling pathways have been identified in the cushions to regulate valve remodeling including members of the Bmp and Egfr pathways. 89 For example, global or endothelial deletion of the heparin-binding epidermal growth factor (HB-EGF) results in normal development but excessive enlargement of the conal cushions due to increased proliferation and prolonged Smad1/5/8 phosphorylation in cushion mesenchyme. 90, 91 Accordingly, this defect is phenocopied in BMP GOF mouse models lacking the Bmp-specific nuclear inhibitor Smad6 92 or the BMP antagonist Noggin. 93 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. MS in preparation). We found that Jag1 can transcriptionally up-regulate HB-EGF, which released in a soluble form associates with the ECM and inhibit mesenchymal cell proliferation through EGFR.94 This suggests that endocardial Notch signaling functions during valve morphogenesis to regulate Egfr and Bmp signaling and thus limit valve cushion proliferation prior to remodeling. Importantly, SLV cusps remodeling may also depend upon appropriate tissue-tissue interactions amongst the SHF and CNC. In addition to the previously described OFT defects, inhibition of Notch in the SHF using the DNMAML transgene resulted in dysmorphic and thickened SLV cusps and aortic regurgitation caused by diminished mesenchymal apoptosis and increased ECM deposition. 95 Given that cardiovascular defects in the Notch SHF mutants are due, in part, to abnormal CNC patterning, CNCs were interpreted to provide instructive signals to orchestrate apoptosis and changes in ECM production during SLV remodeling. 95

SLV malformations are common in humans, including BAV, which occurs in 2-3 percent of the

population, and pulmonic valve stenosis in 10% of all CHD in adults. 96 BAV is usually asymptomatic at birth but increases the risk of disease later in life, including calcification, stenosis and aortopathy. 97 BAV morphology varies between right and left (R-LC) coronary cusp fusion and noncoronary (R-NC) fusion at a ratio of approximately 0.6 to 0.4 (Fig. 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 the OFT cushion whereas the R-L BAV is likely the result of defective OFT septation. 98 Thus far only NOTCH1 has been implicated in the etiology of non-syndromic BAV in human. In a study of two unrelated families, Garg et al. 41 observed mutations in NOTCH1 that segregated with aortic valve disease, particularly with BAV and aortic valve calcification. In addition to BAV, other forms of left ventricular outflow tract obstruction such as coarctation of the aorta and hypoplastic left heart have been related to NOTCH1 polymorpisms as well as mutations in the extracellular domain of NOTCH1. 41, 99 The mechanism of calcific aortic valve disease (CAVD) 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 CAVD in endothelial cells derived from induced human induced pluripotent stem cells extended these findings to multiple epigenetic and transcriptomic targets affected by NOTCH1 haploinsufficiency. 100 Thus hemodynamic shear stress, which protects against calcification, activate anti-osteogenic gene networks and represses pro-inflammatory 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. 100


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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, 101 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. 102-105 Trabeculae increase cardiac output and permit nutrition and oxygen uptake in the embryonic myocardium prior to coronary vascularization 101, 105 and failure in trabeculae formation is embryonic lethal. 106-108 Cells at the tip of the longitudinal axis of the trabeculae are more differentiated than those at the base. 109 As development proceeds, trabeculae extend radially and form a network while also increasing in length. 101 Progressively, the ventricular myocardium differentiates into two distinct layers, the outer, proliferative, compact myocardium and the inner and more differentiated, trabecular myocardium. 109

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,B). These findings suggest that endocardial Notch1 signaling is required for proliferation and differentiation of chamber myocardium. 3, 110, 111 Notch signaling abrogation affects the expression of three signaling pathways required for trabeculation: Bmp10, Nrg1/ErbB and EphrinB2. Bmp10 is expressed in trabecular myocardium where it sustains proliferation. 106 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 trabeculation 107 via activation of ErbB2-4 receptors expressed on myocardium. 112 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. 113 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) while 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. 114 Additional work has shown that during trabeculation the endocardial activity of the peptidyl-prolyl isomerase FKBP12 tightly regulates N1ICD stability. 115 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). Left Ventricular Non-Compaction (LVNC).

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 cells 116 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. 117 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. 105


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Defective trabecular compaction can manifest as a cardiomyopathy termed left ventricular non-compaction (LVNC, OMIM 601493), characterized by the presence of prominent trabeculations separated by deep invaginations in an abnormally thin ventricular wall. 118, 119 LVNC can be a mild disease revealed by a depressed systolic function 120 or can evolve into a serious condition with complications including systemic embolism, malignant arrhythmias, heart failure and sudden death. 121 LVNC is generally transmitted as an autosomal dominant trait, but the underlying molecular mechanisms are still poorly understood. 119 Conditional inactivation in the embryonic mouse myocardium of the Mib1 gene, encoding the E3 ubiquitin ligase Mib1 essential for Notch ligand signaling 14 (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, non-compacted trabeculae, protruding toward the ventricular lumen 4 (Figure 5D,E). 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 non-compacted to compacted myocardium, diagnostic of LVNC in humans. 122 Sequencing of the MIB1 gene in a cohort of 100 LVNC patients (48% familial cases) identified two mutations (V943F and R530X) in two probands. The two 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 two 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 co-immunoprecipitation 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 due to haploinsufficiency caused by insufficient synthesis of WT MIB1 protein; in the case of V943F, this would be due to 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 coronary vessels were down-regulated in mutant embryos. In this regard, Bmp10 was down-regulated in Mib1flox;cTnT-cre mutants, similarly to the situation in endothelial-specific RBPJk mutants 3, indicating that endocardial-myocardial Notch signaling first is crucial 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 is impaired. Our working model suggest that myocardial Mib1 activity 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.


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Endocardial Notch Signaling in Zebrafish Heart Regeneration.

The adult zebrafish heart exhibits a remarkable capacity to regenerate after ventricular resection 123 or cryoinjury-induced myocardial infarction. 124-126 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 dedifferentiation 124-126 as seen in the resection model. 127,

128 Examination of Notch expression in the zebrafish heart after cryoinjury shows a rapid up-regulation of Notch signaling in the endocardium at the injury site (Münch et al. MS submitted), in agreement with reports of Notch reactivation after cardiac resection. 129, 130 Functional and gene expression analysis in the regenerating heart reveals that endocardial Notch activity is required for cardiomyocyte proliferation as reported in the ventricular resection model 129 but also for 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., MS submitted). Disease Modeling.

Mutations in NOTCH signaling components in humans are implicated in CHD (Table 1). The generation and analysis through the years of a great variety of targeted mutant mice harboring standard and/or tissue-specific mutations in several Notch pathway components suggest that a large number of these genes may be involved in CHD (Table 1). This research also illustrates the connection between the fields of developmental biology and pathology. In the simplest situation (Mendelian inheritance) and in order 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 viceversa). For NOTCH, the correspondence between the human and mouse cardiac disease phenotypes has been satisfactory thus far. 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 AGS patients (Table 1). 73, 131 This finding led to the identification of NOTCH2 mutations in a cohort of JAG1 mutation-negative patients with AGS. 68, 132 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 AGS patients. 73

The advent of next-generation sequencing (NGS) technology 133 coupled with efficient DNA

capture 134 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 (GWAS) 135, 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 GWAS 136, 137 and the generation of potentially pathogenic mutations identified by exome sequencing 137 or by any other NGS-related approach.


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Concluding Remarks.

Inthis Review we have described studies that uncover the role of Notch during cardiac patterning, OFT morphogenesis, valve and chamber development and disease. These findings are of fundamental biological interest but also have important implications for understanding the etiology 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 (Tgf2, 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 CAVD; (8) endocardial/endothelial NOTCH1 is required for the activation of anti-osteogenic molecules and repression of pro-inflammatory 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 promote trabecular patterning, maturation and compaction; (10) MIB1 mutations 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 The authors 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 provided English editing. SOURCES OF FUNDING J.L.dlP is funded by grants SAF2013-45543-R, RD12/0042/0005 (RIC) and RD12/0019/0003 (TERCEL) from the Spanish Ministry of Economy and Competitiveness (MINECO) and a grant from the BBVA Foundation for Research in Biomedicine. The CNIC is supported by the MINECO and the Pro-CNIC Foundation.

DISCLOSURES None.


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FIGURE LEGENDS

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 (DSL) motif (light yellow) located in the extracellular domain. Ligand activity is regulated by the ubiquitin ligase Mindbomb1 (Mib1) through ubiquitinylation of the intracellular domain (dark yellow squares). In the signal-receiving cell, the Notch receptor (Notch1-4) 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 two consecutive cleavage events (at S2 and S3 sites, respectively) carried out by an ADAM protease and presenilin, which release the Notch intracellular domain (NICD) that translocates to the nucleus and binds to CSL. In the absence of NICD, CSL associates with co-repressor proteins (Co-R) and HDACs 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.

Figure 2. Key events in cardiac development. A hE14 (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 rightwards (C). At E9.5, formation of the atrio-ventricular canal (AVC) separates the two 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 two paired dorsal aortae that distribute the blood throughout the embryo. Left panel, neural crest cells migrate from the dorsal neural tube, surrounding the aortic arch arteries to the cardiac outflow tract. Middle panel, the neural crest cells that invest the endothelial tubes of the AAAs differentiate into vascular smooth muscle cells. Right panel, 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. Neural crest-derived cells (NCDCs) migrate through the pharyngeal arches and into the OFT to initiate the reorganization of the OFT and formation of semilunar (SL) valves. Epicardial-derived cells (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 four 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-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 etiologies. I, Scheme depicting the thick ventricular walls of the normal adult heart and the thin and trabeculated walls of a non-compacted heart. In all panels the ventral aspect of the heart is shown (anterior to the top). a, atria; avc, atrio-ventricular canal; la, left atrium, lv, left ventricle; oft, outflow tract; rv, right ventricle.

Figure 3. Endocardial Notch activity during cardiac development. A to D, In the E9.5 heart, the ligands Jag1 and Dll4 are expressed in the AVC endocardium (A, A', B, B', D) and in chamber myocardium and endocardium, respectively (A, A'', B, B'',D) while the Notch1 receptor is active in the endocardium (C-C'',D). E-H, At E12.5 Jag1 is strongly expressed in the endocardium lining the OFT endocardial cushions (E,H), while Dll4 expression is reduced compared to E9.5 (F,H), and endocardial N1ICD expression persists (G,H). I,L, At E12.5, Jag1 is strongly expressed in trabecular myocardium (I, arrowhead, L) and


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weakly in the compact myocardium, while 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,P), and N1ICD in chamber endocardium and coronary vessels endothelium (O,P). Abbreviations as in Figure 2.

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 OFT and AVC endocardial cushions where 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) while 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 four 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 Tgf2, 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, SL valves morphogenesis. From E13.5 onwards, once mesenchyme cells occupy the endocardial cushion (EC), cusps 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. Abbreviations as in Figure 2.

Figure 5. Endocardial Notch signaling is essential for chamber development. A Transverse section of an H&E-stained WT E9.5 heart at the level the left ventricle. 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. Modified from 3 and D'Amato et al. unpublished data. D, Episcopic 3D 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 non-compacted trabecular in the mutant heart (E). The asterisk indicates the 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 while the non-proliferative trabecular myocardium is defined by the expression of Anf, Bmp10 and Cx40. Abbreviations as in Figure 2.


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Table 1: Mutations in Notch Pathway Elements Affecting Heart Development in Humans and Mice.

Mouse model Phenotype Defective Endocard. Signaling

Human gene

Disease Mechanism of human

disease

Refs.

Notch1+/- Valve calcification Yes NOTCH1 CAVD, BAV, TOF

HI

41

Notch1-/- Hypoplastic EC & impaired trabeculation

Homozygous lethal (E10.5)

Yes ----- n/a ----- 3,24

Notch1flox/flox; Tie2-Cre

Hypoplastic EC & impaired trabeculation


Yes ----- n/a ----- 3

Notch1flox/flox; Nfatc1pan-Cre

Hypoplastic EC 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 68,73,131,

132

Notch2del1/del1 Myocardial hypoplasia & reduced trabeculation Homozygous lethal

(E10.5)

n.d. NOTCH2 n/a ----- 68,131,132

Notch2flox/ex;Pax3-Cre;

Narrow aortas and pulmonary arteries

n.d. ----- AGS (PAS)

----- 138

Notch2flox/ex;Tag1-Cre

Narrow aortas and pulmonary arteries Neonatal lethality

n.d. ----- AGS (PAS)

----- 138

RBPJk-/- Defective cardiac looping, hypoplastic EC &

impaired trabeculation Homozygous lethal

(E10.5)

Yes RBPJK n/a ----- 3,24

RbpJk+/- Aortic valve sclerosis, stenosis

Yes ----- n/a ----- 139

RBPJkflox/flox;Tie2-Cre Impaired trabeculation Homozygous lethal

(E10.5)

Yes ----- n/a ----- 3

RPJkflox/flox;Dermo1-Cre

Ventricular septal defect Yes

----- n/a ----- 140

Dll4-/- Reduced atrial & ventricular chambers, defective trabeculation

AV malformations Homozygous lethal

(E10.5)

Yes DLL4 n/a ----- 141,142

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


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Jag1flox/flox; Nfatc1-Cre

Hypoplastic EC Homozygous lethal

(E11.5)

Yes ----- n/a ----- 62

Jag1flox/flox; Mef2c-AHF-Cre

AA abnormalities, VSD, ASD

Homozygous neonatal lethal

Yes ----- n/a ----- 78

Jag1flox/flox; Islet1-Cre

AA abnormalities, VSD, ASD

Homozygous neonatal lethal

Yes ----- n/a ----- 78

Mib1flox/flox;cTnT-cre LVNC Yes MIB1 LVNC HI DN

4

Psen1-/- VSD, DORV,PAS Yes PSEN1, PSEN2

Dilated cardiomyopathy

----- 74,143,144

Psen1-/-;Psen2-/- Defective cardiac looping, Homozygous lethal

(E10.5)

n.d. PSEN1, PSEN2

n/a ----- 145

Hey2-/- VSD, ASD, PAS, TOF, TVA, AVV dysfunction

Yes HEY2 n/a ----- 56,58,146,

147 Hey1-/-;Hey2-/- Defective cardiac looping,

hypoplastic EC & impaired trabeculation, homozygous postnatal

lethality

Yes HEY1, HEY2

n/a ----- 57,148

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 ----- 149,75

DNMAML;Pax3-Cre OFT, AA abnormalities, VSD, postnatal lethality

----- MAML n/a ----- 77

DNMAML;Wnt1-Cre OFT, AA abnormalities, postnatal lethality

----- ----- n/a ----- 77

DNMAML;Islet1-Cre OFT, AA abnormalities, VSD, TVA, neonatal

lethality

* ----- n/a ----- 78

DNMAML;Mef2c-AHF-Cre

OFT, AA abnormalities, VSD, neonatal lethality

* ----- n/a ----- 78

Numbflox/flox;Numbl flox/flox;Nkx2.5-Cre

OFT septation defect, DORV, AVSD,

embryonic lethal (E15.5-18.5)

* NUMB, NUMBL

n/a ----- 150

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


Yes ----- n/a ----- 51

R26N1ICD; Nkx2.5-Cre (NGOF)

Hypoplastic EC & thin chamber myocardium, defective trabeculation

embryonic lethal (E11.0)

Yes ----- n/a ----- 51


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R26N1ICD; cTnT-Cre (NGOF)

Hypoplastic EC & 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, aortic arch; ASD, atrial septum defect; AGS, Alagille syndrome; AV, atrio-ventricular; AVSD, atrio-ventricular septum defect; AVV, atrio-ventricular valve; BAV, bicuspid aortic valve; CAVD, calcific aortic valve disease; DN, dominant negative; DORV, double outlet right ventricle; EC; endocardial cushion; HGOF, Hey gain-of-function; HI, haploinsufficiency; LVNC, left ventricular non-compaction; n.a., not-applicable; NGOF, Notch gain-of-function; n.d., not-determined; OA, overriding aorta; PAS, pulmonary artery stenosis; RVHP, right ventricular hypoplasia; TOF, tetralogy of Fallot; TVA, tricuspid valve atresia; VSD, ventricular septal defect; *, hypothetical


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Guillermo de Luxán, Gaetano D'Amato, Donal MacGrogan and Jose Luis de la PompaEndocardial Notch Signaling in Cardiac Development and Disease

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2015 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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