心脏流出道单细胞转录状态参考图谱的构建及其在流出道畸形先心病的发病、发展机理研究中的应用

Cardiac outflow tract during embryogenesis is a major hotspot for congenital heart diseases. Outflow tract malformations, represented by the transposition of the great arteries (TGA), are characterized by high diagnostic frequency and mortality. However, the mechanism for pathogenesis and progression remains largely unknown. Cell transcriptomic state reference atlas of normal development constructed by single-cell RNA-seq not only can help to understand the normal development process, but also provides a control for studying diseased states. In a pilot study, we obtained single-cell transcriptomic data for 55,611 cells of normal developing outflow tract, and constructed a preliminary reference atlas. It is known that the derivatives of cardiac neural crest cells (CNCCs) play a critical role in outflow tract remodeling and septation. In this project, we plan to further construct a transcriptomic state reference map specifically for cells derived from CNCCs using single-cell RNA-seq. Based on the updated reference map for normal developing outflow tract (as a control), we will comparatively study the transcriptomic state changes in outflow tract cells of high concentration retinoic acid-induced TGA mouse model. We seek to identify cell subpopulations and cell type-specific expression regulations that response to retinoic acid signaling. We also try to elucidate the effects of retinoic acid signaling on the migration and differentiation of CNCCs. We hope that this project will put novel insights into the cellular and molecular mechanism for pathogenesis and progression of outflow tract malformations at a single-cell level.

发育中的心脏流出道是先心病发病的一个主要热点。以大动脉转位为代表的一类称为流出道畸形的先心病具有发病比例高、致死率高的特点。而目前对流出道畸形的发病、发展机制很大程度上依旧未知。利用单细胞转录组数据构建正常发育中的细胞转录状态参考图谱不仅能够系统理解正常发育过程，而且能为流出道畸形的研究打下参考基础。在前期研究中我们获得了55,611个正常发育中的小鼠流出道细胞的单细胞转录组数据，并初步构建了转录状态参考图谱。已知心脏神经脊细胞后代在流出道的重构中扮演重要角色。本项目计划进一步构建针对心脏神经脊细胞后代的参考图谱，并以升级后的图谱作为“对照”，通过单细胞转录组比较研究视黄酸诱导大动脉转位的发生过程中流出道细胞转录状态的变化，揭示对视黄酸信号响应的细胞亚群和细胞类型特异性的表达调控，探讨视黄酸信号对心脏神经脊细胞的迁移和分化的影响。以期在单细胞水平上为流出道畸形的细胞、分子机理提供新见解。

发育中的心脏流出道是先心病发病的一个主要热点。以大动脉转位为代表的一类称为流出道畸形的先心病具有发病比例高、致死率高的特点。然而目前人们对流出道畸形先心病发生、发展的细胞分子机制的认识依据十分有限。我们在前期对发育中的小鼠心脏流程道单细胞图谱绘制的基础上（2019年发表在Cell Reports杂志），本项目首先利用大规模单细胞转录组分析阐明了对流出道发育贡献很大的心脏神经脊来源细胞后代在心脏发育过程中的转录状态异质性、分化轨迹和调控网络（相关结果发表在细胞生物学著名期刊EMBO Reports杂志）。其次，我们通过整合单细胞转录组与全外显子组分析，首次发现了纤毛基因突变在散发大动脉转位的发病过程中可能扮演有重要角色（发表在心血管领域著名期刊Circulation Research杂志）。再次，我们对高浓度视黄酸诱导的大动脉转位小鼠模型进行了单细胞转录组分析，发现在流出道发育早期阶段，心肌细胞和心内膜内皮细胞的内在改变可能是大动脉转位发生的潜在原因（结果整理中）。此外项目执行过程中还衍生出一些基于单细胞组学数据挖掘的新的课题思路。截止项目结题，共发表5篇中科院1区研究论文。