Delta-like ligand-4 regulates Notch-mediated maturation of second heart field progenitor-derived pharyngeal arterial endothelial cells.

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.

Concomitant genetic defects potentiate the adverse impact of prenatal alcohol exposure on cardiac outflow tract maturation.

BACKGROUND: Prenatal alcohol exposure (PAE) is associated with an increased incidence of congenital heart defects (CHD), in particular outflow tract (OFT) defects. However, the variability in the incidence of CHD following PAE has not been fully explored. We hypothesize that a concomitant, relevant genetic defect would potentiate the adverse effect of PAE and partially explain the variability of PAE-induced CHD incidence. METHODS: The OFT is formed by the second heart field (SHF). Our PAE model consisted of two intraperitoneal injections (3 g/kg, separated by 6 hr) of 30% ethanol on E6.5 during SHF specification. The impact of genetic defects was studied by SHF-specific loss of Delta-like ligand 4 (Dll4), fibroblast growth factor 8 (Fgf8) and Islet1. RESULTS: Acute PAE alone significantly increased CHD incidence (4% vs. 26%, p = .015) with a particular increase in OFT alignment defects, viz., double outlet right ventricle (0 vs. 9%, p = .02). In embryos with a SHF genetic defect, acute PAE significantly increased CHD incidence (14 vs. 63%, p < .001), including double outlet right ventricle (6 vs. 50%, p < .001) compared to controls. PAE (p = .01) and heterozygous loss of Dll4 (p = .04) were found to independently contribute to CHD incidence, while neither Islet1 nor Fgf8 defects were found to be significant. CONCLUSIONS: Our model recapitulates the increased incidence of OFT alignment defects seen in the clinic due to PAE. The presence of a concomitant SHF genetic mutation increases the incidence of PAE-related OFT defects. An apparent synergistic interaction between PAE and the loss of DLL4-mediated Notch signaling in OFT alignment requires further analysis.

Murine Model of Cardiac Defects Observed in Adams-Oliver Syndrome Driven by Delta-Like Ligand-4 Haploinsufficiency.

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.

Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression.

The role played by the Notch pathway in cardiac progenitor cell biology remains to be elucidated. Delta-like ligand 4 (Dll4), the arterial-specific Notch ligand, is expressed by second heart field (SHF) progenitors at time-points that are crucial in SHF biology. Dll4-mediated Notch signaling is required for maintaining an adequate pool of SHF progenitors, such that Dll4 knockout results in a reduction in proliferation and an increase in apoptosis. A reduced SHF progenitor pool leads to an underdeveloped right ventricle (RV) and outflow tract (OFT). In its most severe form, there is severe RV hypoplasia and poorly developed OFT resulting in early embryonic lethality. In its milder form, the OFT is foreshortened and misaligned, resulting in a double outlet right ventricle. Dll4-mediated Notch signaling maintains Fgf8 expression by transcriptional regulation at the promoter level. Combined heterozygous knockout of Dll4 and Fgf8 demonstrates genetic synergy in OFT alignment. Exogenous supplemental Fgf8 rescues proliferation in Dll4 mutants in ex-vivo culture. Our results establish a novel role for Dll4-mediated Notch signaling in SHF biology. More broadly, our model provides a platform for understanding oligogenic inheritance that results in clinically relevant OFT malformations.