Bicuspid aortic valve (BAV) is the commonest congenital cardiac malformation in man (Hoffman and Kaplan, 2002). Together with stenosis of the aortic and pulmonary valves, BAV commonly accompanies other congenital cardiovascular malformations, for example, ventricular septal defects, double outlet right ventricle, hypoplastic left heart syndrome and aortic coarctation (Siu and Silversides, 2010). BAV is also associated with late onset aortopathy (Tadros et al., 2009). These associations may reflect common developmental origins and/or abnormal haemodynamic forces, but little is known specifically about arterial (semilunar) valve development and mechanisms have been extrapolated from tricuspid and mitral (atrioventricular) valve development (de Vlaming et al., 2012). Similarly, the pathogenesis of stenotic, dysplastic or bicuspid arterial valves remains obscure, although fusion of adjacent leaflets in fetal life, resulting in the presence of a raphe, is presumed to cause many cases of BAV.

Over the past 20 years we have developed a good understanding of how the outflow tract develops. Cells derived from the second heart field (SHF) make the major contribution to the outflow tract walls, forming myocardium in the proximal ventricular part, smooth muscle cells in the distal arterial walls, and also the inner endocardial lining (Kelly et al., 2014). In the early unseptated outflow tract, peristaltic contraction propels blood forward and retrograde flow is prevented by occlusion of the heart tube lumen by voluminous cardiac jelly interposed between endothelium and contracting myocardium (Nomura-Kitabayashi et al., 2009). As the outflow tract lengthens, the cardiac jelly becomes localised to discrete areas, endocardial cushions, which are the anlagen of the mature valve leaflets (de Vlaming et al., 2012; Phillips et al., 2013). These initially acellular endocardial cushions become populated with mesenchymal cells that give rise to the valve interstitial cells. A major source of cells in both atrioventricular and arterial valves is epithelial or endothelial to mesenchymal transformation (EMT or EndMT; [Markwald et al., 1977; Markwald et al., 1978; de Vlaming et al., 2012]). In addition, the main outflow tract cushions, that fuse to separate aortic and pulmonary blood flow and also give rise to the arterial valve leaflets, receive a major contribution of neural crest cells (NCC) that have migrated through the pharyngeal arches (Kirby et al., 1983; Waldo et al., 1999; Jiang et al., 2000; Jain et al., 2011; Phillips et al., 2013). In contrast few NCC are seen in the atrioventricular valves. Smaller swellings located between the main superior and inferior outflow tract cushions, commonly called intercalated cushions (IC), give rise to the remaining two leaflets (Kramer, 1942; Anderson et al., 2012; Sizarov et al., 2012; Yang et al., 2013). We have previously shown that NCC are essential for positioning the main outflow tract endocardial cushions and patterning the arterial valve leaflets, but make only a minor contribution to these IC (Phillips et al., 2013). More recently, we have also shown that the arterial roots (Figure 1A) that connect the ventricles with the great arteries and house the arterial valves, have a complex origin from both SHF-derived cells and NCC (Richardson et al., 2018). However, the contribution of each lineage to the different valve leaflet primordia, and how these relationships affect normal and abnormal leaflet patterning including BAV, remains obscure.

Figure 1 with 1 supplement see all

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This study demonstrates how the arterial valve leaflets form and defines the origins of the cells that populate these leaflets. We show that despite the mature arterial valve leaflets appearing indistinguishable, the IC have an entirely distinct origin and underpinning developmental mechanisms from the major cushions, originating from a novel, direct SHF contribution that is independent of endMT.

We have shown that the formation of the arterial valves, whilst encompassing some processes that are similar to the atrioventricular valves, is uniquely different, by virtue of the large influx of NCC and the dominant role played by the SHF. The central four valve leaflets, formed as the outflow tract septates, are filled by NCC and SHF cells secondarily derived through endocardium and transformed into valve interstitial cell precursors by EndMT. In contrast, the intercalated valve structures are not cardiac jelly-derived endocardial cushions, but instead differentiate directly from (Mef2c-AHF-Cre+;Tnnt2-Cre+) SHF-derived outflow wall; Figure 9). These cells retain signatures of previous cardiac troponin promoter activation, although they are not, and never have been, of myocardial phenotype. As the ICVS mature, they switch on valve leaflet markers such as Sox9 and Versican, without utilising EndMT as a mechanism or expressing typical mesenchymal markers. Ultimately, however, they appear indistinguishable from the valve leaflets derived from the main outflow cushions. Specifically, we find no evidence of trans-differentiation of either cardiomyocytes or SMC to the interstitial cells within the ICVS, the concomitant reduction of Isl1 and increase in Sox9 protein levels within the ICVS instead indicating a mechanism of direct differentiation of SHF progenitors into valve cells.

Figure 9

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These studies show that three lineages make significant contributions to the arterial valve leaflets; NCC, EDC and a novel population of SHF progenitors cells that transiently activate the Tnnt2-Cre driver, but differentiate directly into valve interstitial cells without passing through the endocardium nor myocardial lineages. This novel population makes the major contribution to the anterior and posterior leaflets derived from the ICVS, and also significant contributions to the left and right leaflets, derived from the main cushions (Figure 9). Although a technical limitation of these Cre-based studies is the inability to track both SHF and NCC lineages concomitantly, the absolute numbers of cells counted, and the proportions of each, suggest that any other lineages can only comprise a minor component (Figure 9). Previous studies have suggested that the possibility of trans-differentiation of cardiomyocytes in the distal outflow (Ya et al., 1998). These studies were performed before knowledge of the SHF and the now accepted concept that cells are added to the distal poles of the outflow tract. Our data strongly argue against trans-differentiation as a mechanism for formation of the ICVS as although the Tnnt2-Cre+ cells activated the cTnT promoter, we never saw co-expression of cardiomyocyte markers in the forming ICVS and could identify a few Tnnt2-Cre+/cTnI- cells even at E9.5, before the ICVS form. As well as making the major contribution to the forming ICVS, these Tnnt2-Cre+ lineage cells contribute cells to the postnatal valve leaflets although detailed phenotyping and analysis of the fate of these cells is beyond the scope of this manuscript.

Cre lineage tracing identifies cells and their descendants that currently, or have ever, expressed the promoter/enhancer element used to drive Cre protein-based recombination of a reporter gene. It is evident from this study that to interpret such data it is essential to track protein expression of the Cre-driven promoter product. In the original description of the Tnnt2-Cre driver (Jiao et al., 2003), it was stated that the rat cTnT promoter construct drove cardiomyocyte-specific expression up to E10.5, but its expression pattern later in gestation was not described. It has also been reported that cTnT is expressed in some SMC and can modulate their contraction (Wang et al., 2001; Kajioka et al., 2012). From data presented here it is clear that Tnnt2-Cre labels arterial SMC, as well as the ICVS. However, as we could not find evidence of cTnT protein expression in these latter cell types, even when we looked at the earliest stages, it is likely that transient activation of the promoter occurred in undifferentiated, potentially multipotent, SHF progenitors within the distal outflow. Analysis of this phenomenon in relation to the SMC is the subject of further study in our laboratory. With respect to the ICVS, rather than becoming cardiomyocytes, Tnnt2-Cre labeled cells activated Sox9 expression and differentiated to valve interstitial cells, although this occurred at E11.5, approximately a day later than the more proximal cells within the wall differentiated to cardiomyocytes.

Our data are generally in agreement with and build on the data of Sizarov et al (Sizarov et al., 2012). However, these authors did not observe Sox9 staining within the cells of the ICVS of approximately stage-matched human embryos, although they did see Isl1 labelling. They also suggested that the ‘columns’ they described were mesenchymal, although our data show that, at least in the early stages, they are epithelial. Moreover, we did not see expression of classical mesenchymal markers such as N-cadherin, αSMA and Fsp1, although the ICVS did express elastin, which is found in mature valve leaflets. The differences between these studies are unclear, but it may be that they reflect the transient expression of some of these markers and/or slight differences in staging and maturation of the ICVS between mouse and human. The delay in differentiation of cells within the ICVS may at least partially explain the activation of the Tnnt2 promoter in these cells, as they may be poised, but stalled, for differentiation. The presence of Tnnt2-Cre+ lineage cells in the ICVS of the mouse may be a valuable tool to investigate other genes involved in ICVS development, as it allows deletion in the directly-differentiating SHF population in the ICVS, without affecting NCC or EDC. For example, Mib1 is a ubiquitin ligase required in signalling cells for Jag activity. Here, and in a previous publication (Captur et al., 2016), arterial valve defects were observed in Mib1;Tnnt2-Cre mutants, although initially the reason for this was unclear. Our data suggest that these defects likely arise because Tnnt2-Cre is activated in the valve primordia, deleting Mib1 and thus inactivating Jag1/Jag2-Notch signalling. This prevents the ICVS, and to a lesser extent the main cushions, from forming properly thus resulting in abnormal leaflets in the mutant embryos.

EndMT is a crucial process for typical endocardial cushions, but not for ICVS, where the major process is development from SHF cells that directly differentiate into valve interstitial cells in situ. In contrast, there is minor deposition of ECM and Sox9+ cells in the sub-endothelial compartment of the forming ICVS, indicative of a small contribution by EndMT to the ICVS in this region. Notch1 together with Dll4, plays a critical early role during EndMT and Notch signalling is known to be vital for normal development of the main endocardial cushions. Deletion of both Notch1 and Dll4 (Limbourg et al., 2005), result in the early death of the embryos, by E10.5, precluding examination for ICVS formation and thus making it difficult to exclude EndMT from a crucial role in formation of the ICVS. However, evidence from the knockout of Brg1 in endothelium using Nfatc1-Cre (Akerberg et al., 2015) provides evidence that ICVS are not dependent on EndMT as both anterior and posterior arterial valves leaflets appeared well formed in this mutant.

Our mouse data, in keeping with those of Sizarov who studied human embryos (Sizarov et al., 2012), suggest that the ICVS form as expansions of compacted cells within the wall of the distal OFT from cells in continuity with the epithelial SHF. These condensations form at the proximal extent of the ‘fishmouth’ of non-myocardial distal outflow wall (Anderson et al., 2012) as seen in Figure 1O, likely reflecting the asymmetrical addition of SHF cells and the rotation of the outflow wall (Scherptong et al., 2012; Bajolle et al., 2006). These cell condensations then separate from the surrounding wall, forming discrete structures that then undergo differentiation to typical valve cells. Thus, these ICVS cells appear to share an origin and be juxtaposed with the myocardial component of the outflow wall. Indeed, both the myocardial and the ICVS cells are Tnnt2-Cre+ suggesting a close relationship between the two populations. It is of note that Jag1 was abundant in the ICVS, which do not utilise EndMT/EMT, and is also abundantly expressed in the outflow tract myocardium. Although Jag1 is also essential for valve development, its role in EndMT is controversial, with some authors suggesting it is important (Wang et al., 2013), but others suggesting it is not required for EndMT but plays a later role in the remodelling phases of the main cushion-derived heart valves (MacGrogan et al., 2016). From our data, and those of others (High et al., 2007), it seems likely that it is Notch1 and/or Notch2 that is the main Notch receptor in the ICVS. Notch1ICD was found in the distal outflow wall and the ICVS, but only transiently, and it was largely down-regulated by E11.5. Notch2 was expressed throughout the ICVS and until E11.5, although as we could not obtain an antibody for the active form of this protein, we cannot be sure that signalling was underway throughout this period. This transient expression within the ICVS is in contrast to the endocardium overlying the main cushions, where both Notch1 and Notch2 were maintained until E12.5 and beyond (Del Monte et al., 2007). Notch signalling has been shown to be important for the differentiation of cardiac progenitor cells, with its down-regulation crucial for differentiation to cardiomyocytes (Nemir et al., 2006; Kwon et al., 2009) and for activation of Sox9 in cartilage precursors (Mead and Yutzey, 2009). Thus, the presence and then subsequent removal of Notch1 signalling may be essential in the ICVS for their differentiation to valve interstitial cells. In support of the idea that Notch-Jag signalling is playing an important role in the development of the ICVS, the deletion of Jag1/Jag2 using Tnnt2-Cre resulted in abnormal development of the arterial valve leaflets in 9/11 double mutants examined, including BAV without raphe. Specifically, in 5/11 cases the phenotype was absence or hypoplasia of the non-coronary leaflet that is derived from the ICVS. Although the Jag1/2;Tnnt2-Cre mice have a range of defects affecting the ventricular myocardium (D'Amato et al., 2016), and so valve defects secondary to the ventricular defects cannot strictly be ruled out, it seems more likely that the reduction/loss of the non-coronary leaflet results from the deletion of Jag expression specifically in the ICVS. The redundancy of Jag1 and Jag2 is intriguing and is reminiscent of ventricular wall development (D'Amato et al., 2016). Although Jag1 was expressed in the core of the ICVS, Jag2 was restricted to the adluminal cells, close to N1ICD-expressing cells, suggesting that there may be some ligand-specific functional specialization. Further studies will be necessary to delineate these likely complex interactions.

The Vangl2;Isl1-Cre and Tbx1 mutant mouse lines, both of which have abnormalities associated with limited addition of SHF cells to the outflow, have abnormal valve leaflets that result from failure to form or mature the ICVS; a mechanism that has not previously been described. In the Vangl2 mutant, premature differentiation of SHF to cardiomyocytes, as occurs earlier in the aortic sac (Ramsbottom et al., 2014), may reduce the availability of SHF-derived cells to form Sox9+ valve interstitial cells and may underlie the reduction in the size of the ICVS and ultimately lead to BAV. The reduction in Notch1ICD and Jag1 in 2 of 3 Vangl2^f/f;Isl1-Cre embryos suggests that Notch signalling may be playing a role in this process and be regulated by Vangl2, either directly or indirectly. Notably, the defects were more common in the aortic valve (as they are in the Notch signalling pathway mutants) and although the reasons for this are uncertain, aortic (but never pulmonary) hypoplasia is common in the naturally occurring Vangl2 mutant, loop-tail (Henderson et al., 2001). This suggests that it is the SHF that contributes to the aorta that is preferentially affected in this mutant. Premature loss of progenitor phenotype and differentiation of cardiomyocytes leading to a marked reduction of SHF-derived subpulmonary myocardium has been reported in the Tbx1 mutant mouse (Vitelli et al., 2010). In addition, absence of the anterior leaflet of the pulmonary valve (derived from the pulmonary ICVS) has also been reported (Théveniau-Ruissy et al., 2008). Our analyses extend these data to show that a discrete pulmonary ICVS is never formed in the Tbx1^-/- and highlights similarities in the ICVS phenotype between these and the Vangl2;Isl1-Cre mutants. Moreover, the role of both Vangl2 and Tbx1 in regulating the epithelial properties of the SHF (Ramsbottom et al., 2014; Francou et al., 2014), which form the myocardium and also appear to form the ICVS, suggests that the epithelial phenotype of these cells, at least at early stages of their development, may be crucial. Indeed, it raises the possibility that there is an intrinsic relationship between the loss of epithelial properties of the wall and differentiation of the cells within it to cardiomyocytes. These differences between the affected valve observed between Vangl2 and Tbx1 mutants likely reflect inherent differences in the patterning of the outflow tract, further reflecting subdomains within the SHF (Vitelli et al., 2010; Scherptong et al., 2012). BAV has been described in a number of mouse mutants. Notably, in several cases (including following knockout of Alk2, a BMP receptor (Thomas et al., 2012), and Slit/Robo (Mommersteeg et al., 2015) signalling) the anterior/non-coronary leaflets specifically failed to form properly. This, therefore may be a common mechanism underlying BAV (without raphe) formation, at least in mice, and may occur when there is disruption of the SHF as we see in the Vangl2;Isl1-Cre and Tbx1 mutants. Raphe are infrequently described in mouse mutants with BAV, although this may be because most mouse mutants are described during fetal life when the leaflets remain unsculpted and thus before a true raphe would be apparent. Although it is not possible to demonstrate the mechanisms of arterial valve development using human embryos it is important to note that at comparable stages of development the appearances of human and mouse valve primordia are very similar and importantly, the expression pattern of Isl1, marking SHF progenitors, is identical. Together our data suggest that loss of SHF progenitors, either because of a failure of their formation or their premature differentiation, may be a common cause of BAV and can explain the common association between this and other intra-cardiac heart malformations. As such, BAV formed in this way may link to the subtype of BAV patterns without raphe, as occurs in 5–10% of BAV cases (Sievers and Schmidtke, 2007; Buchner et al., 2010). It will be important to analyse genomic data from patients with this BAV phenotype, searching in particular in SHF and Jag-Notch pathways.

Our murine and human data show differences between the structure of the aortic and pulmonary ICVS as they develop, that could have relevance to the common incidence of BAV compared to bicuspid pulmonary valve. The aortic ICVS develops more proximally and is not surrounded by a myocardial cuff as it develops, compared with the pulmonary ICVS, which is more distal and is completely surrounded by cardiomyocytes. These differences have also been reported recently in a histological examination of human embryos (Milos et al., 2017). Moreover, the pulmonary ICVS has a lesser contribution from NCC than does the aortic, and based on the differences we see between Vangl2 and Tbx1 mutants, may have different genetic regulation. The ICVS also express tropoelastin during their formation, whereas the main cushions do not, suggesting that these differences in cell lineage between the forming leaflets are reflected by expression of specific proteins. These differences are likely to have morphogenetic impacts on the developing aortic and pulmonary ICVS, which might reflect in their differential predisposition to pathology. Although bicuspid pulmonary valve is rare as an isolated finding in humans, it is common in association with tetralogy of Fallot (Chacko et al., 2017). Thus, defects in the SHF that result in failure to develop one or other ICVS might be relevant to a high proportion of human valve abnormalities.

All data are included in the manuscript and supplementary files.

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Author details

1. Lorriane Eley

   Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

2. Ahlam MS Alqahtani

   Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Donal MacGrogan

   1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
   2. Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares, Instituto de Salud Carlos III, Madrid, Spain

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Rachel V Richardson

   Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Lindsay Murphy

   Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Alejandro Salguero-Jimenez

   1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
   2. Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares, Instituto de Salud Carlos III, Madrid, Spain

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Marcos Sintes Rodriguez San Pedro

   Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Shindi Tiurma

   Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Lauren McCutcheon

   Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

10. Adam Gilmore

    Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

    Contribution

    Investigation

    Competing interests

    No competing interests declared

11. José Luis de La Pompa

    1. Intercellular Signalling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
    2. Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares, Instituto de Salud Carlos III, Madrid, Spain

    Contribution

    Resources, Supervision, Funding acquisition, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6761-7265

12. Bill Chaudhry

    Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2833-8882

13. Deborah J Henderson

    Institute of Genetic Medicine, Cardiovascular Research Centre, Newcastle University, Newcastle upon Tyne, United Kingdom

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    deborah.henderson@ncl.ac.uk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2705-5998

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