Data set for thesis submission entitled "Characterization of cardiac defects associated with Vinculin deletion in cardiac neural crest"

Vinculin (Vcl) is an adaptor protein of adherens junctions and focal adhesions. Cardiac
neural crest cell (CNCC) is a key population of progenitor cells regulating the cardiac outflow
tract (OFT) septation and valvulogenesis. Neurocristopathy patients carrying a loss-of-function
mutation in VCL presented various cardiac defects. A mouse model of NCC-specific 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.

Vinculin（Vcl）为粘着连接和焦点粘附的适配蛋白。心脏神经嵴细胞（CNCC）是调节心脏输出道（OFT）分隔和瓣膜形成的祖细胞关键种群。携带VCL功能缺失突变的神经节病（Neurocristopathy）患者表现出各种心脏缺陷。Vcl-cKO（NCC特异性敲除Vcl的小鼠模型）完美地模拟了患者中的心脏缺陷，并表现出超增殖的半月瓣。本研究旨在通过Vcl-cKO表征NCC介导的瓣膜形成的分子机制。对E13.5胚胎心脏中CNCC祖细胞进行的单细胞转录组分析揭示了在突变细胞中转化生长因子-β（TGF-β）信号严重中断。更有趣的是，随后的免疫组化分析（IHC）进一步发现TGF-β信号在介导CNCC-和内皮衍生瓣膜间质细胞（VICs）之间的通讯及其在瓣膜形成过程中的激活中发挥新的作用。TGF-β信号的缺陷中断了CNCC衍生VICs的激活，导致心肌化延迟和瓣膜重塑失败。TGF-β信号中断在E10.5时观察到，中断了心内膜垫的形成，对E10.5至E15.5期间瓣膜间质细胞中TGF-β激活的时序跟踪进一步揭示了在半月瓣形成过程中存在两波TGF-β信号激活，分别参与不同的发育过程。在突变VICs中，TGF-β信号中断被发现从E13.5至E15.5出现延迟上调，结合细胞增殖和凋亡实验，可能表明Vcl-cKO突变中观察到的超增殖半月瓣与CNCC-VICs异常增殖有关，这种增殖与TGF-β信号激活的延迟上调相关。总之，研究发现Vcl通过调节CNCC中的TGF-β信号在介导半月瓣形成中发挥关键作用。CNCC作为TGF-β信号中心，对EndoMT和VIC的激活和分化起着至关重要的作用。