Aortic sac
A orta Pulmonary ^ o rta Pulm onary ^ o r ta Pulmonary trunk
NCC
trunk
SCx
tru n k
E10.5
E11.5
E12.5
E13.5
Doi sal
Right-
Left
The Cardiac Neural Crest
The neural crest cells are a population of epithelial cells that delaminate from the length of the tips of the neural folds, migrate throughout the embryo, and have a variety o f essential functions during embryogenesis. Neural crest cells from all axial levels give rise to glia, melanocytes and sensory neurons (LaBonne and Bronner-Fraser,
1998). In addition, other structures to which their progeny give rise are determined by their original position along the length of the neural tube. For example, cranially- derived neural crest cells give rise to components of the craniofacial skeleton and teeth (Chai et al., 2000), and neural crest cells that delaminate from the mid-otic placode to the third somite give rise to the tunica media of the branchial arch arteries and the aortic sac, and also to structures in the outflow tract of the heart. It is this latter group which are described in the current section.
Pre-migratory neural crest cells from chick embryos can be replaced with pre- migratory quail neural crest cells and the embryos allowed to develop in ovo. The donor quail cells can then be distinguished from host chick cells on the basis of histology following Feulgen and Rossenbeck staining, or by using quail specific
antibodies (Waldo et a l, 1998). This type of chimeric study led to the identification of the specific group of neural crest cells that migrate from between the mid-otic placode and somite 3 and colonise the aortic sac and heart via the 3"^^, 4^^ and 6* branchial arch arteries, and are known as the cardiac neural crest (Le Douarin and Teillet, 1974; Le Lievre and Le Douarin, 1975; Kirby et a l, 1983). These studies showed that the cardiac neural crest cells populate the aortopulmonary septum of the aortic sac (Figure 1.14 a). They also migrate, via the aortic sac, whose wall they contribute cells to, into the outflow tract cushions. Once in the cushions, many of the neural crest cells organise into a distinct structure which resembles an upside down “Y” or “U” (Waldo et a l,
1998; Jiang et a l, 2000), the two prongs extending into the cushions and the base being 75
at the aortopulmonary septum (Figure 1.14 b). This structure has been called the (aorticopulmonary) septation complex (Waldo et a l, 1998). The structure then moves proximally, although how far is unclear (Figure 1.14 c) (Poelmann et a l, 1998; Waldo
et a l, 1998). Ablation of pre-migratory chick cardiac neural crest cells results in
outflow tract defects such as in a common arterial trunk, in which both the outflow tract and the aortic sac fail to septate (Kirby et a l, 1983).
Recent work using retroviral labelling of chick neural crest cells with a lacZ
reporter gene has confirmed these results (Poelmann et a l, 1998). However these studies have also suggested that there is a group of apoptosis-prone neural crest cells from more caudal regions of the neural tube that migrate to the venous pole of the heart (Poelmann and Gittenberger-de Groot, 1999). The exact function of these neural crest cells is unknown; some of them appear to colonise the atrioventricular cushions, while many of them are distributed in the chick in a manner that is reminiscent of the chick cardiac conduction system, suggesting that they may be important for its development (Poelmann and Gittenberger-de Groot, 1999).
Studies in which the reporter gene lacZ has been driven by promoters of genes expressed in neural crest cells have shown that neural crest cells are found in the mouse aortic sac and outflow tract, from E10.5, in a comparable distribution to that seen in the chick (Waldo et a l, 1999; Epstein et a/.,2000; Jiang et a l, 2000). Importantly,
however, these studies show no evidence for cardiac neural crest cells entering the venous pole or colonising the atrioventricular cushions of the mouse heart. Further evidence for a role for neural crest cells in mammalian systems comes from the study of mouse mutants. In the splotch 2 / / mutant mouse, for example, a mutation in the Paired box-3 (Pax-3) transcription factor results in defective neural crest cell migration (Epstein et a l, 1991; Conway et a l, 1997b) and common arterial trunk (Franz, 1989; Conway et a l, 1997a). This phenotype is very similar to that seen in the chick
following neural crest cell ablation. Occasionally, as occurs around 5% of the time in
splotch mutant mice with the 2H allele, the distal outflow tract does septate, but the proximal does not (Conway et a l, 1997a). This leads to the outflow tract exiting solely from the right ventricle and the malformation double outlet right ventricle. It has been inferred from, this and the distribution of the neural crest cells, that both proximal and distal outflow tract septation requires a contribution from the neural crest cells. The role of neural crest cells with relation to outflow tract septation is described in further detail in the following section, and an association of cardiac neural crest cells with apoptosis in the heart and their fate is described in Section 1.3.5.
In rodent models, there is strong evidence that cardiac neural crest cells express the structural protein a-smooth muscle actin once they enter the aortic sac and outflow tract. Alpha-smooth muscle actin is expressed in exactly the same regions of the aortic sac and the outflow tract, particularly in the septation complex, in which transgenic analysis has shown neural crest cells to be present, but not in the atrioventricular cushions (Ya et a l, 1998a; Ya et a l, 1998b; Waller, III et a l, 2000; Waldo et a l, 1999; Conway et a l, 2000; Epstein et a l, 2000; Jiang et a l, 2000). Hence a-smooth muscle actin provides a marker for early colonising neural crest cells in the outflow tract.
Cardiac neural crest cells and outflow tract septation
On arrival at the aortic sac, at E l0.5 in the mouse (Jiang et a l, 2000), the cardiac neural crest cells populate and help form the aortopulmonary septum. This septum grows from the dorsal wall of the aortic sac between the entrances to the 4^ and branchial arch arteries (Figure 1.14a; Ya et a l, 1998a). Growth of the septum, which eventually fuses with the outflow tract cushions at E l 1.5, results in separation of the aorta and pulmonary trunks (Figure 1.14 b; Fananpazir and Kaufman, 1988). As described above, failure of proper neural crest cell colonisation of the septum leads to
its underdevelopment, which in turn leads to failure of aortic sac septation (Kirby et a l,
1983; Conway et a l, 1997). Once septated, the common wall (i.e. the septum) between the aorta and the pulmonary trunk will itself divide and become fibrous in order to form the apposing walls of the aorta and pulmonary trunk (Figures 1.14 c and d)(Waldo et a l, 1998). However, the mechanism by which this occurs is not known.
The mechanism of the initial formation of the outflow tract cushions has already been described in Section 1.2.4. At E10.5, as the aortic sac is septating, neural crest cells invade the cushions by means of the walls of the aortic sac (Figure 1.14a; Jiang et a l, 2000). At E l 1.5, the distal end of the cushions fuses with the aortopulmonary septum (Figure 1.14 b). During E l 1.5, the neural crest cells present in the mass of the cushions become organised into the septation complex, described above (Figure 1.14 b). The base of this structure then appears to move proximally, and as it does so, is
associated with fusion of the outflow tract cushions (Figures 1.14 b-c; Y â e t a l, 1998; Jiang et a l, 2000). This observation, and the fact that in experiments where neural crest cell migration is impaired, the outflow tract cushions fail to fuse, has led to the
suggestion that the septation complex is necessary for proper fusion of at least the distal outflow tract cushions (Poelmann et a l, 1998; Waldo et a l, 1998), although exactly how this occurs remains unclear.
By E13.5 in the mouse, the proximal outflow tract cushions have completely fused with each other, the interventricular septum and the superior atrioventricular cushions, completely dividing the outflow tract, and so separating the pulmonary and systemic circulations, and marking the end of septation (Figure 1.14 d and compare Figures 1.15 a and b). While our understanding of the septation complex provides some explanation of how the distal cushions fuse, it does not explain fusion of the more proximal outflow tract cushions. This is because the base of the aortopulmonary septum has never been demonstrated to extend to the most proximal part of the outflow
Figure 1.15 Outflow tra ct septation II
Figures a) and b) are sketches o f the heart made using transverse sections at El 2.5 and E l3.5 respectively. The views are ventral and a “window” has been made into the heart to enable outflow tract septation to be viewed. The arrow in a) shows an
interventricular communication.
a) The proximal outflow tract cushions have almost completely fused, however, there is still a communication between the left and right ventricles (arrow), b) The proximal outflow tract cushions have completely fused together, and with the interventricular septum and the superior atrioventricular cushion, completing septation by closing the communication shown in a).
Ao =aorta
SV OFTC = sinistro-ventral outflow tract cushion IC = inferior
LA = left atrium LV = let ventricle
DDOFTC = dextro-dorsal outflow tract cushion PT = pulmonary trunk
RA = right atrium RV = right ventricle