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Mesodermal progenitors in the second heart field (SHF) express Delta-like-ligand 4 (Dll4) that regulates Notch-mediated proliferation. As cells of SHF lineage mature to assume endocardial and myocardial cell fates, we have shown that Dll4 expression is lost, and the subsequent expression of another Notch ligand Jagged1 regulates Notch-mediated maturation events in the developing heart. A subset of SHF progenitors also matures to form the pharyngeal arch artery (PAA) endothelium. Dll4 was originally identified as an arterial endothelial-specific Notch ligand that plays an important role in blood vessel maturation, but its role in aortic arch maturation has not been studied to date secondary to the early lethality observed in Dll4 knockout mice. We show that, unlike in SHF-derived endocardium and myocardium, Dll4 expression persists in SHF-derived arterial endothelial cells. Using SHF-specific conditional deletion of Dll4, we demonstrate that as SHF cells transition from their progenitor state to an endothelial fate, Dll4-mediated Notch signalling switches from providing proliferative to maturation cues. Dll4 expression maintains arterial identity in the PAAs and plays a critical role in the maturation and re-organization of the 4th pharyngeal arch artery, in particular. Haploinsufficiency of Dll4 in SHF leads to highly penetrant aortic arch artery abnormalities, similar to those observed in the clinic, primarily resulting from aberrant reorganization of bilateral 4th pharyngeal arch arteries. Hence, we show that cells of SHF lineage that assume an arterial endothelial fate continue to express Dll4 and the resulting Dll4-mediated Notch signalling transitions from an early proliferative to a later maturation role during aortic arch development.

Heterozygous loss-of-function mutation in Delta-like ligand-4 (Dll4) is an important cause of Adams-Oliver syndrome (AOS). Cardiac defects, in particular outflow tract (OFT) alignment defects, are observed in about one-fourth of patients with this syndrome. The mechanism underlying this genotype-phenotype correlation has not yet been established. Dll4-mediated Notch signaling is known to play a crucial role in second heart field (SHF) progenitor cell proliferation. We hypothesized that the depletion of the SHF progenitor pool of cells due to partial loss of Dll4 is responsible for the OFT alignment defects seen in AOS. To demonstrate this, we studied Dll4 expression by murine SHF progenitor cells around E9.5, a crucial time-point in SHF biology. We used SHF-specific (Islet1-Cre) conditional knockout of Dll4 to bypass the early embryonic lethality seen in global Dll4 heterozygotes. Dll4-mediated Notch signaling is critically required for SHF proliferation such that Dll4 knockout results in a 33% reduction in proliferation and a fourfold increase in apoptosis in SHF cells, leading to a 56% decline in the size of the SHF progenitor pool. A reduction in SHF cells available for incorporation into the developing heart leads to underdevelopment of the SHF-derived right ventricle and OFT. Similar to the clinical syndrome, 32% of SHF-specific Dll4 heterozygotes demonstrate foreshortened and misaligned OFT, resulting in a double outlet right ventricle. Our murine model provides a molecular mechanism to explain the cardiac defects observed in AOS and establishes a novel clinical role for Dll4-mediated Notch signaling in SHF progenitor biology.

Introduction: Wnt5a is a known regulator of planar cell polarity (PCP) signaling in second heart field (SHF) progenitor cells. It is generally believed that Wnt5a ligands are secreted by the SHF; however, SHF-specific conditional deletion of Wnt5a does not recapitulate outflow tract (OFT) defects observed in global Wnt5a mutants. Hypothesis: Given the proximity and interaction between neural crest cells (NCC) and cranial SHF that contributes to the developing OFT, we hypothesize that NCC may serve as an additional source of Wnt5a for SHF progenitors. Methods: Wnt5a was conditionally deleted in the neural crest using transgenic Wnt1-cre mice. Embryos were harvested from control and mutant litter mates and immunofluoresence, in situ hybridization, and hematoxylin-eosin stains were performed on histologic sections using standard techniques. India ink injections were performed to evaluate pharyngeal arch artery and outflow tract morphology in whole mount embryos. Results: Wnt1-cre driven conditional deletion of Wnt5a in NCC did not impact NCC survival or migration into the developing OFT. However, SHF cells in mutant E10.5 embryos showed altered PCP signaling with reduced phalloidin and laminin expression. The resulting loss in polarized directional movement of the SHF led to reduced incorporation into and elongation of the developing OFT. The shortened OFT was mal-aligned resulting in fully penetrant double outlet right ventricle (DORV) at E14.5. In addition, maturation of pharyngeal arch arteries was also impaired such that all mutants express pharyngeal arch artery defects, including aortic arch abnormalities and aberrant right subclavian artery. In contrast, there was no observed effect on PCP in the more caudal SHF cells, and none of the mutant embryos had inflow tract or venous pole defects. Conclusions: Our results demonstrate that NCC are a novel source of Wnt5a. NCC-derived Wnt5a is critically required to regulate PCP signals in the most cranial SHF regulating the development of the OFT and pharyngeal arch arteries.