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Congenital heart defects (CHDs) affecting the cardiac outflow tract (OFT) constitute a significant cause of morbidity and mortality. The OFT develops from migratory cell populations which include the cardiac neural crest cells (cNCCs) and secondary heart field (SHF) derived myocardium and endocardium. The related transcription factors HAND1 and HAND2 have been implicated in human CHDs involving the OFT. Although Hand1 is expressed within the OFT, Hand1 NCC-specific conditional knockout mice (H1CKOs) are viable. Here we show that these H1CKOs present a low penetrance of OFT phenotypes, whereas SHF-specific Hand1 ablation does not reveal any cardiac phenotypes. Further, HAND1 and HAND2 appear functionally redundant within the cNCCs, as a reduction/ablation of Hand2 on an NCC-specific H1CKO background causes pronounced OFT defects. Double conditional Hand1 and Hand2 NCC knockouts exhibit persistent truncus arteriosus (PTA) with 100% penetrance. NCC lineage-tracing and Sema3c in situ mRNA expression reveal that Sema3c-expressing cells are mis-localized, resulting in a malformed septal bridge within the OFTs of H1CKO;H2CKO embryos. Interestingly, Hand1 and Hand2 also genetically interact within the SHF, as SHF H1CKOs on a heterozygous Hand2 background exhibit Ventricular Septal Defects (VSDs) with incomplete penetrance. Previously, we identified a BMP, HAND2, and GATA-dependent Hand1 OFT enhancer sufficient to drive reporter gene expression within the nascent OFT and aorta. Using these transcription inputs as a probe, we identify a novel Hand2 OFT enhancer, suggesting that a conserved BMP-GATA dependent mechanism transcriptionally regulates both HAND factors. These findings support the hypothesis that HAND factors interpret BMP signaling within the cNCCs to cooperatively coordinate OFT morphogenesis.

Neural crest cells (NCCs) are physically responsible for craniofacial skeleton formation, pharyngeal arch artery remodeling and cardiac outflow tract septation during vertebrate development. Cdc42 (cell division cycle 42) is a Rho family small GTP-binding protein that works as a molecular switch to regulate cytoskeleton remodeling and the establishment of cell polarity. To investigate the role of Cdc42 in NCCs during embryonic development, we deleted Cdc42 in NCCs by crossing Cdc42 flox mice with Wnt1-cre mice. We found that the inactivation of Cdc42 in NCCs caused embryonic lethality with craniofacial deformities and cardiovascular developmental defects. Specifically, Cdc42 NCC knockout embryos showed fully penetrant cleft lips and short snouts. Alcian Blue and Alizarin Red staining of the cranium exhibited an unfused nasal capsule and palatine in the mutant embryos. India ink intracardiac injection analysis displayed a spectrum of cardiovascular developmental defects, including persistent truncus arteriosus, hypomorphic pulmonary arteries, interrupted aortic arches, and right-sided aortic arches. To explore the underlying mechanisms of Cdc42 in the formation of the great blood vessels, we generated Wnt1Cre-Cdc42-Rosa26 reporter mice. By beta-galactosidase staining, a subpopulation of Cdc42-null NCCs was observed halting in their migration midway from the pharyngeal arches to the conotruncal cushions. Phalloidin staining revealed dispersed, shorter and disoriented stress fibers in Cdc42-null NCCs. Finally, we demonstrated that the inactivation of Cdc42 in NCCs impaired bone morphogenetic protein 2 (BMP2)-induced NCC cytoskeleton remodeling and migration. In summary, our results demonstrate that Cdc42 plays an essential role in NCC migration, and inactivation of Cdc42 in NCCs impairs craniofacial and cardiovascular development in mice.

DiGeorge syndrome (DGS), characterized genetically by a deletion within chromosome 22q11.2, is associated with a constellation of congenital heart defects. DiGeorge critical region 8 (Dgcr8), a gene that maps to the common deletion region of DGS, encodes a double stranded RNA-binding protein that is essential for miRNA biogenesis. To address the potential contribution of Dgcr8 insufficiency to cardiovascular development, we have inactivated Dgcr8 in cardiac neural crest cells (cNCCs). Dgcr8 mutants displayed a wide spectrum of malformations, including persistent truncus arteriosus (PTA) and ventricular septal defect (VSD). Interestingly, Dgcr8-null cNCCs that properly migrated into the cardiac outflow tract (OFT), proliferate normally and differentiate into vascular smooth muscle cells. However, loss of Dgcr8 causes a significant portion of the cNCCs to undergo apoptosis, causing a decrease in the pool of progenitors required for OFT remodeling. Our data uncover a new role of Dgcr8 in cardiovascular morphogenesis, plausibly as part of transmission mechanism for FGF-dependent survival cue for migrating cNCCs.

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., and Meyers, E.N. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.); however, subsequent reports describe only a single outflow tract, leaving the phenotype and its developmental mechanism unclear. We hypothesized that the phenotype that occurs in response to Shh knockdown is pulmonary atresia and is directly related to the abnormal development of the secondary heart field. We found that Shh was expressed by the pharyngeal endoderm adjacent to the secondary heart field and that its receptor Ptc2 was expressed in a gradient in the secondary heart field, with the most robust expression in the caudal secondary heart field, closest to the Shh expression. In vitro culture of secondary heart field with the hedgehog inhibitor cyclopamine significantly reduced proliferation. In ovo, cyclopamine treatment before the secondary heart field adds to the outflow tract reduced proliferation only in the caudal secondary heart field, which coincided with the region of high Ptc2 expression. After outflow tract septation should occur, embryos treated with cyclopamine exhibited pulmonary atresia, pulmonary stenosis, and persistent truncus arteriosus. In hearts with pulmonary atresia, cardiac neural crest-derived cells, which form the outflow tract septum, migrated into the outflow tract and formed a septum. However, this septum divided the outflow tract into two unequal sized vessels and effectively closed off the pulmonary outlet. These experiments show that Shh is necessary for secondary heart field proliferation, which is required for normal pulmonary trunk formation, and that embryos with pulmonary atresia have an outflow tract septum.

Msx1 and Msx2 are highly conserved, Nk-related homeodomain transcription factors that are essential for a variety of tissue–tissue interactions during vertebrate organogenesis. Here we show that combined deficiencies of Msx1 and Msx2 cause conotruncal anomalies associated with malalignment of the cardiac outflow tract (OFT). Msx1 and Msx2 play dual roles in outflow tract morphogenesis by both protecting secondary heart field (SHF) precursors against apoptosis and inhibiting excessive proliferation of cardiac neural crest, endothelial and myocardial cells in the conotruncal cushions. During incorporation of SHF precursors into the OFT myocardium, ectopic apoptosis in the Msx1−/−; Msx2−/− mutant SHF is associated with reduced expression of Hand1 and Hand2, which from work on Hand1 and Hand2 mutants may be functionally important in the inhibition of apoptosis in Msx1/2 mutants. Later during aorticopulmonary septation, excessive proliferation in the OFT cushion mesenchyme and myocardium of Msx1−/−; Msx2−/− mutants is associated with premature down-regulation of p27KIP1, an inhibitor of cyclin-dependent kinases. Diminished accretion of SHF precursors to the elongating OFT myocardium and excessive accumulation of mesenchymal cells in the conotruncal cushions may work together to perturb the rotation of the truncus arteriosus, leading to OFT malalignment defects including double-outlet right ventricle, overriding aorta and pulmonary stenosis.

Morphogenesis of the cardiac arterial pole is dependent on addition of myocardium and smooth muscle from the secondary heart field and septation by cardiac neural crest cells. Cardiac neural crest ablation results in persistent truncus arteriosus and failure of addition of myocardium from the secondary heart field leading to malalignment of the arterial pole with the ventricles. Previously, we have shown that elevated FGF signaling after neural crest ablation causes depressed Ca2+ transients in the primary heart tube. We hypothesized that neural crest ablation results in elevated FGF8 signaling in the caudal pharynx that disrupts secondary heart field development. In this study, we show that FGF8 signaling is elevated in the caudal pharynx after cardiac neural crest ablation. In addition, treatment of cardiac neural crest-ablated embryos with FGF8b blocking antibody or an FGF receptor blocker rescues secondary heart field myocardial development in a time- and dose-dependent manner. Interestingly, reduction of FGF8 signaling in normal embryos disrupts myocardial secondary heart field development, resulting in arterial pole malalignment. These results indicate that the secondary heart field myocardium is particularly sensitive to FGF8 signaling for normal conotruncal development, and further, that cardiac neural crest cells modulate FGF8 signaling in the caudal pharynx.

Previous studies have demonstrated that TGFbeta induces a smooth muscle fate in primary neural crest cells in culture. By crossing a conditional allele of the type II TGFbeta receptor with the neural crest-specific Wnt1cre transgene, we have addressed the in vivo requirement for TGFbeta signaling in smooth muscle specification and differentiation. We find that elimination of the TGFbeta receptor does not alter neural crest cell specification to a smooth muscle fate in the cranial or cardiac domains, and that a smooth muscle fate is not realized by trunk neural crest cells in either control or mutant embryos. Instead, mutant embryos exhibit with complete penetrance two very specific and mechanistically distinct cardiovascular malformations--persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA-B). Pharyngeal organ defects such as those seen in models of DiGeorge syndrome were not observed, arguing against an early perturbation of the cardiac neural crest cell lineage. We infer that TGFbeta is an essential morphogenic signal for the neural crest cell lineage in specific aspects of cardiovascular development, although one that is not required for smooth muscle differentiation.

The cardiac neural crest contains ectomesenchymal and neural anlagen that are necessary for normal heart development. It is not known whether other regions of the neural crest are capable of supporting normal heart development. In the experiments reported herein, quail donor embryos provided cardiac, trunk, or mesencephalic neural crest to replace or add to the chick host cardiac neural crest. Neither trunk nor mesencephalic neural crest was capable of generating ectomesenchyme competent to effect truncal septation. Addition of mesencephalic neural crest resulted in a high incidence of persistent truncus arteriosus, suggesting that ectomesenchyme derived from the mesencephalic region interferes with ectomesenchyme derived from the cardiac neural crest. Derivatives from the trunk neural crest, on the other hand, did not result in abnormal development of the truncal septum. While mesencephalic neural crest seeded the cardiac ganglia with both neurons and supporting cells, this capability was limited in the trunk neural crest to the more mature regions. These studies indicate a predetermination of the ectomesenchymal derivatives of the cranial neural crest and a possible competition of neural anlagen to form neurons and supporting cells in the cardiac ganglia.

Neural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT). Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severe OFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCs lacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. This contributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressing cardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may result from abnormal differentiation or defective recruitment of differentiating SHF cells into OFT. Alterations in Bmp4, Sema3C, and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt the communications among different cell populations. Such disruptions can further affect the recruitment of NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septation failure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affecting the positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cell autonomous effects on their survival and non-cell autonomous effects on coordinating the movement of multiple cell lineages in the positioning and the remodeling of the OFT.

Heart outflow tract septation in mouse embryos carrying mutations in retinoic acid receptor genes fails with complete penetrance. In this mutant background, ectopic TGFβ signaling in the distal outflow tract is responsible for septation failure, but it was uncertain what tissue was responsive to ectopic TGFβ and why this response interfered with septation. By combining RAR gene mutation with tissue-specific Cre drivers and a conditional type II TGFβ receptor (Tgfbr2) allele, we determined that ectopic activation of TGFβ signaling in the endocardium is responsible for septation defects. Ectopic TGFβ signaling results in ectopic mesenchymal transformation of the endocardium and thereby in improperly constituted distal OFT cushions. Our analysis highlights the interactions between myocardium, endocardium, and neural crest cells in outflow tract morphogenesis, and demonstrates the requirement for proper TGFβ signaling in outflow tract cushion organization and septation.

Systemic loss-of-function studies have demonstrated that Pax3 transcription factor expression is essential for dorsal neural tube, early neural crest and muscle cell lineage morphogenesis. Cardiac neural crest cells participate in both remodeling of the pharyngeal arch arteries and outflow tract septation during heart development, but the lineage specific role of Pax3 in neural crest function has not yet been determined. To gain insight into the requirement of Pax3 within the neural crest, we conditionally deleted Pax3 in both the premigratory and migratory neural crest populations via Wnt1-Cre and Ap2α-Cre and via P0-Cre in only the migratory neural crest, and compared these phenotypes to the pulmonary atresia phenotype observed following the systemic loss of Pax3. Surprisingly, using Wnt1-Cre deletion there are no resultant heart defects despite the loss of Pax3 from the premigratory and migratory neural crest. In contrast, earlier premigratory and migratory Ap2α-Cre mediated deletion resulted in double outlet right ventricle alignment heart defects. In order to assess the tissue-specific contribution of neural crest to heart development, genetic ablation of neural crest lineage using a Wnt1-Cre-activated diphtheria toxin fragment-A cell-killing system was employed. Significantly, ablation of Wnt1-Cre-expressing neural crest cells resulted in fully penetrant persistent truncus arteriosus malformations. Combined, the data show that Pax3 is essential for early neural crest progenitor formation, but is not required for subsequent cardiac neural crest progeny morphogenesis involving their migration to the heart or septation of the outflow tract.