Characterization of cardiac defects associated with vinculin deletion in cardiac neural crest cells

Abstract

Vinculin (Vcl) is an adaptor protein of adherens junctions and focal adhesions. Cardiac neural crest cells (CNCC) constitute a key population of progenitor cells regulating the cardiac outflow tract (OFT) septation and valvulogenesis. Neurocristopathy patients carrying a lossof- function mutation in VCL presented various cardiac defects. A mouse model of NCCspecific knockout of Vcl (Vcl-cKO) nicely phenocopied the cardiac defects as seen in the patients and exhibited hyperplastic semilunar valves. Here, we aimed to characterize the molecular mechanisms underlying the NCC-mediated valvulogenesis using Vcl-cKO. Single cell transcriptomic analysis of CNCC progenies in E13.5 embryonic hearts revealed that transforming growth factor-b (TGF-b) signaling is severely interrupted in the mutant cells. More intriguingly, subsequent immunohistochemistry analysis (IHC) further discovered a novel role of TGF-b signal in mediating the crosstalk between CNCC- and endothelial- derived valvular interstitial cells (VICs) and their activation during valvulogenesis. Defective TGF-b signal interrupted the activation of CNCC-derived VICs, leading to retarded myocardialization and failure in valve remodeling. While TGF-b signaling disruption was observed at E10.5 interrupting the formation of endocardial cushion, temporal tracking of TGF-b activation in valvular interstitial cells from E10.5 to E15.5 further revealed two waves of TGF-b signaling activation in semilunar valvulogenesis, contributing to separate developmental processes. The disruption of TGF-b signaling was found with a delayed upregulation from E13.5 to E15.5 in mutant VICs. Together with cell proliferation and apoptosis assays, it might suggest the hyperplastic SLV observed in Vcl-cKO mutant was associated with aberrant proliferation of CNCC-VICs through delayed TGF-b signaling activation. In summary, it was found that Vcl plays a crucial role in mediating semilunar valvulogenesis through TGF-b signaling regulation in CNCCs. CNCCs contributed essentially as a TGF-b signaling hub to EndoMT and VIC activation and differentiation.