Our results demonstrate what we believe is a novel role for neural crest cells in semilunar valve leaflet maturation. During normal cardiogenesis, migrating neural crest and second heart field cells are in close proximity in the ventral pharynx and endocardial cushions (Figure , K and L). Our data suggest that instructive cues are exchanged between these cell fields and these cues are necessary for semilunar valve maturation. Specifically, we provide evidence that signals from the neural crest populating the outflow endocardial cushions mediate alterations in extracellular matrix production and promote apoptosis that characterizes late gestation semilunar valve leaflet remodeling (Figure ). Deletion of Pax3 results in abnormal neural crest patterning and bicuspid-like valve leaflets, which are characterized by an increase in extracellular matrix and deficiency of late-gestation apoptosis. Notch inhibition in the second heart field results in abnormal neural crest cell patterning, and these mutants also display similarly dysmorphic semilunar valve leaflets, though we cannot exclude a direct effect on second heart field–derived mesenchyme. These results provide evidence that semilunar valve leaflet remodeling is dependent upon appropriate tissue-tissue interactions amongst the second heart field, neural crest, and valve mesenchyme. Though all our mutants display outflow tract defects, our data, in accord with others, suggest that semilunar valve leaflet maturation occurs late in gestation, while septation and patterning of the outflow tract occur at earlier time points (15
). Nonetheless, we cannot exclude the possibility that the valve abnormalities we detect are secondary to or dependent upon the outflow defects.
Model depicting the role of neural crest in semilunar valve development.
Several studies have shown an association between NOTCH1
mutations and the presence of a bicuspid aortic valve. Garg et al. identified 2 families with multiple generations with congenital cardiac defects including bicuspid valve disease that were associated with mutations in NOTCH1
, including R1108X resulting in a premature stop codon and H1505del resulting in a frameshift mutation and truncated protein (21
). Other studies have identified other NOTCH1
mutations associated with sporadic forms of bicuspid aortic valve, including those with thoracic aortic aneurysms (22
). These studies did not address the specific tissue in which Notch functions during semilunar valve maturation. Notch signaling regulates endothelial-mesenchymal transformation during endocardial cushion formation (43
). However, our data suggest that abnormalities of Notch signaling in neural crest (9
) or second heart field can also contribute to the development of abnormal semilunar valves. Our previous studies have shown that inhibition of Notch in the second heart field impairs Fgf8 signaling (15
). Interestingly, engineered deficiencies in Fgf8 can also result in bicuspid aortic valve and vascular smooth muscle abnormalities of the great arteries (44
). Fgf8 expressed by second heart field mesoderm is likely to function in an autocrine fashion (46
) and the signals that mediate crosstalk between the second heart field and neural crest remain to be fully elucidated. Further studies investigating the modulators of Notch and Fgf8 signaling in syndromic and nonsyndromic cases of bicuspid aortic valve will be of interest.
Multiple types of aortic arch malformations are associated with abnormal semilunar valves, including bicuspid aortic valve. The association between bicuspid aortic valve and aortic dissection and aneurysm is well documented, and interestingly, studies have shown that up to 60% of patients with aortic coarctation have a concomitant bicuspid aortic valve (28
). In addition, multiple cases of carotid and vertebral artery dissection in patients with bicuspid aortic valve have been reported (26
). In some cases, vascular abnormalities may be secondary to abnormal hemodynamics created by a faulty aortic valve. However, multiple studies have demonstrated aortic dissections and aneurysms in patients without hypertension, hemodynamic perturbations, or with only mildly abnormal valves, suggesting an intrinsic defect in the aorta of these patients (26
). Interestingly, evidence of cystic medial necrosis, a process in which aortic vascular smooth muscle undergoes apoptosis, has been found in patients before dilation of the aorta is clinically noted (28
). In addition, evidence of fragmented elastic fibers with greater distance between fibers has been reported (31
). The contribution of neural crest to the smooth muscle of the ascending aorta and aortic arch is well documented (7
), and our neural crest and Notch mutants reveal disorganized aortic intimal layers with increased distance between cells (Supplemental Figure 4 and ref. 45
). To the best of our knowledge, our data provide the first experimental evidence unifying the developmental mechanisms underlying semilunar valve and aortic arch abnormalities.
TGF-β signaling has been extensively studied in the setting of aortic aneurysm formation, Marfan syndrome, and related disorders (50
). Marfan syndrome is a disease characterized by mutations in FIBRILLIN1
, which usually sequesters TFG-β. Without this modulation, an increase of TGF-β signaling manifests as aortic root dissection, skeletal overgrowth, pulmonary emphysema, and ocular lens dislocation, the classic signs of Marfan syndrome (50
). Loeys-Dietz syndrome is an autosomal dominant syndrome closely related to Marfan syndrome, characterized by gain-of-function mutations in the genes encoding the type I or II TGF-β receptor, arterial tortuosity, arterial dissections and aneurysms, bicuspid aortic valve, and various craniofacial abnormalities (50
). Importantly, the aortas of Marfan syndrome patients and patients with bicuspid aortic valve and aortic aneurysm demonstrate loss of elastic fiber architecture and abnormal distribution of collagen and other extracellular matrix proteins (53
). Vascular specimens from Loeys-Dietz patients and nonsyndromic cases of patients with bicuspid aortic valve and aortic aneurysm demonstrate increased TGF-β signaling. Reduction of TGF-β signaling through the administration of neutralizing antibodies or chemical antagonists can attenuate the phenotypes (51
). Notch and TGF-β signaling have been shown to cooperate to promote vascular smooth muscle differentiation (57
), although we were unable to detect increases in phospho-Smad2/3 in our Notch and neural crest mutants (data not shown). Further studies will be needed to elucidate the role of TGF-β signaling in the tissue-tissue interactions that we have described during aortic arch and semilunar valve remodeling.
As the neural crest migrates through the pharyngeal mesenchyme, it is in close proximity to the second heart field, a likely location for signaling between these 2 cell populations (Figure , F and G). Bmp4 is robustly expressed in the 2 columns of cardiac neural crest as they enter the outflow tract and populate the endocardial cushions (15
) and is a mediator of apoptosis in other tissues (58
). Bmp4 expression in cardiac neural crest is diminished upon Notch inhibition in the second heart field (15
). Hence, Bmp4 is an attractive candidate as a mediator of apoptotic signaling from neural crest that can respond to signals from the second heart field (15
). Indeed, Bmp4 is required for proper aortic arch remodeling and semilunar valve development (59
). However, deletion of Bmp4
in the neural crest (using Wnt1-cre) does not result in abnormal semilunar valves (data not shown). Msx signaling has been shown to be an important regulator of neural crest apoptosis (61
), and Msx
genes can function downstream of Bmp signaling. However, we have not observed consistent changes in Msx1
expression in late gestation Notch mutant valves (data not shown). Finally, MMP signaling has been the focus of intense investigation in its role in bicuspid aortic valve and aortic aneurysm formation and progression. Specifically, MMP-2 and MMP-9 levels are altered in in vitro and ex vivo studies of bicuspid aortic valve and aneurysms (53
). However, it must be elucidated whether abnormal levels of MMP-2 or MMP-9 are causal in the phenotype or a marker of disease. In addition, deciphering the spatial and temporal expression of MMP signaling during semilunar valve development will be of great interest. The identification of the neural crest–derived apoptotic signal or signals that are required for semilunar valve remodeling will be the focus of future studies, as will the characterization of signals responsible for altered extracellular matrix production.
In summary, our data suggest that neural crest provides an instructive signal for remodeling of the semilunar valves and highlight the importance of tissue-tissue interactions among second heart field, neural crest, and endocardial cushion mesenchyme. The experimental demonstration of a role for neural crest in the pathophysiology of congenital semilunar valve disorders provides a developmental mechanism to explain the association of aortic and pulmonary valve defects with vascular abnormalities of the aortic arch.