A study of the roles of vinculin in the cardiac neural crest cell development and the associated cardiovascular anomalies

Abstract

Vinculin (Vcl) is a key adaptor protein at the focal adhesion (FA) serving as a mechanical sensor to mediate various cellular processes. Our group has previously demonstrated that a loss-of-function mutation in VCL interrupts the development of neural crest cells (NCCs), leading to ventricular septal defect (VSD) in humans. In this study, I further delineated how Vcl regulates the development of cardiac NCC (CNCC) and contributes to heart development using NCC-specific Vcl knockout (VclKOWnt1-Cre) mice.

VclKOWnt1-Cre mutants died few hours after birth due to severe heart failures. All mutants at E18.5 presented with VSD, 90% of them had persistent truncus arteriosus (PTA), and 60% showed interrupted aortic arch (IAA). In addition, hyperplastic semilunar valves were found in these mutants, suggesting that the formation of valves was interrupted.

Cardiac defects of the mutants are primarily caused by the problems of CNCCs in migration and differentiation. At E10.5, delayed population of the 4th pharyngeal arch arteries (PAA) and the outflow tract (OFT) by CNCCs was observed in the mutants. Some mutant cells failed to differentiate into smooth muscle cells (SMC), and that interrupted the subsequent formation of the aortic arch and caused the incomplete septation of aorta and pulmonary trunk, leading to IAA and PTA, respectively. At E13.5, the mutant CNCCs in the valve primordium also failed to induce the myocardialization to guide myocardial cells to invade endocardial cushions (EC), such that the adjacent mesenchymal cells remained largely undifferentiated, and excessive mesenchymal cells were left within the valves. The valves were unable to undergo valve remodeling and remained bulbous and swollen at E17.5. Defective myocardialization was also observed in the proximal OFT, such that the formation of the membranous ventricular septum was perturbed, leading to VSD. Therefore, Vcl is required for mediating the migration and differentiation of CNCCs to drive the PAA patterning, OFT septation, semilunar valve formation, and ventricular septum closure.

To further reveal the molecular mechanisms underlying these cellular processes, single-cell transcriptomes of CNCCs from E13.5 control and mutants were analyzed. Although the mutant CNCCs could give rise to both neural (neurons and glia) and non-neural (mesenchyme and SMC) derivatives, the proportion of SMC was reduced in the mutants. These SMCs exhibited unique gene expression profiles with predominant involvement in the GO terms “OFT morphogenesis” and “cellular responses to the transforming growth factor beta (Tgf-β) stimulus”. Importantly, the mutant SMC showed elevated expression of Sox9 and Tbx20, accompanied by down-regulation of the Notch ligand, Jag1, and that likely interrupted the subsequent valve remodeling. In E13.5 VclKOWnt1-Cre embryonic hearts, consistently, fewer numbers of CNCC progenies exhibited activated Tgf-β signaling (pSmad2/3+) when compared to the control. Moreover, significantly larger numbers of Sox9+ and Tbx20+ CNCCs were found in ECs of E13.5 VclKOWnt1-Cre hearts. In summary, all these data suggest that Vcl-mediated mechanosensing and Tgf-β signaling are required to drive CNCCs to differentiate towards the SMC lineage and for inducing the Jag1/Notch pathway for the subsequent valve remodeling.