Background
A considerable percentage of cardiac birth defects is caused by a failure in normal migration, differentiation or patterning of the cardiac neural crest (CNC). This subset of pluripotent neural crest stem cells forms in the dorsal aspect of the neural tube at the level of the mid-otic placode to the third somite [1]. Subsequently cardiac neural crest cells (CNCCs) delaminate, undergo a phenotypic transformation from an epithelial to mesenchymal cell type, and migrate latero-ventrally into the 3rd, 4th and 6th pharyngeal arch arteries (PAAs), where they contribute to the formation of the smooth muscle cell layer of endothelial structures derived from the aortic arch arteries [1-3]. A subset of CNCCs continues to migrate deeper into the aortic sac to form the aortico-pulmonary septum; a vital structure, which separates the pulmonary trunk from the aorta [4].
An indispensable role of CNCCs in the development of the cardiac outflow tract was originally demonstrated by pioneering studies of Kirby and coworkers [1], who showed that ablation of the CNC in the chick led to severe outflow tract (OFT) defects including persistent truncus arteriosus (PTA), mispatterning of the great vessels and outflow tract mal-alignments [5]. Early migratory CNCCs have been shown to retain a considerable degree of plasticity and their fate is largely controlled by instructional signals from local environments into which NCCs migrate [6]. Several recent studies have indicated that members of the TGF-β superfamily, i.e., TGF-βs and BMPs are likely candidates to provide some of these signals. Mice deficient in TGF-β2 display fourth aortic arch artery defects [7], while neural crest cell specific abrogation of TGF-β type II receptor (Tgfbr2) results in interruption of the aortic arch and PTA [8,9]. BMPs 6 and -7 are required for proper formation of the outflow tract cushions [10], while BMP type II receptor is needed for proper development of the conotruncal ridges [11]. Moreover, neural crest-specific deletion of the BMP type I receptors Alk2 and Alk3 has been shown to lead to defective aortico-pulmonary septation, among other cardiac defects [12,13].
TGF-β subfamily ligands signal via a receptor complex composed of two type II receptors and two type I receptors [14,15]. Ligand binding leads to phosphorylation and activation of type I receptors, which, in turn, phosphorylate and activate a specific set of downstream signaling molecules called Smads. In general terms, TGF-βs bind to the TGF-β type II receptor (TGFβRII) and TGF-β type I receptor (ALK5) activating TGF-β Smads (2 and 3), while BMPs bind to the BMP type II receptor and type I receptors ALK2, -3, or 6, activating BMP Smads (1, 5 and 8). However, it is likely that these signaling interactions are more complex in vivo, possibly allowing formation of heterotetrameric complexes composed of different type II and type I receptors [16]. In addition, recent studies have identified novel TGF-β-related ligands, which can bind to entire different combinations of receptors. For instance, growth and differentiation factors (GDFs) 8 and 9 can bind to Activin type II receptor and ALK5 to activate TGF-β Smads [17,18]. Therefore, we hypothesized that deletion of Alk5 in a specific cell lineage should reveal phenotypes which cannot be seen in comparable mutants lacking Tgfbr2. Indeed, we recently showed that neural crest cell specific Alk5 mutants display a unique spectrum of craniofacial developmental defects, e.g., cleft snout and severe mandibular hypoplasia [19]; these phenotypes were not seen in corresponding Tgfbr2 mutants [20]. To determine, whether ALK5 would also mediate unique non-redundant signaling events in cardiac neural crest cells, we focused on cardiac and pharyngeal phenotypes of mouse embryos lacking Alk5 specifically in neural crest cells. We discovered that in Alk5/Wnt1-Cre mutants, pharyngeal organs (thymus and parathyroid) fail to migrate appropriately. Moreover, the mutant embryos display severe aortic sac and pharyngeal arch artery defects, and failed aortico-pulmonary septation leading to PTA. Our data further suggest that at least some of these abnormal detected phenotypes result from a dramatic increase in apoptosis of postmigratory cardiac neural crest cells. These phenotypes differ remarkably from those seen in corresponding Tgfbr2 mutants, suggesting that ALK5 mediates a wider spectrum of signaling events than its classical binding partner TGFβRII in cardiac neural crest cells during cardiac and pharyngeal development.