Single cell evaluation of endocardial HAND2 gene regulatory networks reveals critical HAND2 dependent pathways impacting cardiac morphogenesis.

The basic-helix-loop-helix transcription factor HAND2 plays a critical role in endocardium to myocardium signaling during cardiac morphogenesis through its role within the NOTCH signaling pathway. Hand2 endocardial deletion (H2CKOs) results in embryonic lethality by E13.5 due to tricuspid atresia (TA) or double inlet left ventricle (DILV) with accompanying intraventricular septum defects, multiple septa, and hypo-trabeculated ventricles. In addition, H2CKOs also exhibit an increased density of coronary lumens. In order to understand the underlying regulatory mechanisms that cause these phenotypes, we undertook a single cell transcriptome analysis of E11.5 H2CKOs hearts. Hand2fx/+Nfatc1Cre males were crossed with Hand2fx/fxR262RmTmG/mTmG females to generate embryos at E11.5. Cre status determined by presence or absence of GFP fluorescence. Rapid genotyping used to adetermine the status of Hand2 conditional allele. Whole hearts from control and H2CKO embryos were dissociated for single cell sequencing.碱性螺旋-环-螺旋转录因子HAND2（basic-helix-loop-helix transcription factor HAND2）在心脏形态发生过程中，通过参与NOTCH信号通路（NOTCH signaling pathway）介导的心内膜向心肌信号转导发挥关键作用。Hand2心内膜特异性敲除模型（H2CKOs）可导致胚胎于胚胎发育第13.5天（E13.5）死亡，其表型包括三尖瓣闭锁（tricuspid atresia, TA）或左心室双入口（double inlet left ventricle, DILV），同时伴随室间隔缺损、多分隔形成及心室肌小梁发育不全。此外，H2CKOs胚胎还表现出冠状管腔密度升高的异常特征。为阐明引发上述异常表型的潜在调控机制，本研究对胚胎发育第11.5天（E11.5）的H2CKOs心脏开展了单细胞转录组分析。实验中，将基因型为Hand2fx/+Nfatc1Cre的雄性小鼠与Hand2fx/fxR262RmTmG/mTmG的雌性小鼠杂交，以获取E11.5天的胚胎；通过GFP荧光的有无判定Cre重组酶的表达状态，并通过快速基因分型确定Hand2条件性等位基因的状态；收集对照组与H2CKO胚胎的完整心脏，经解离处理后用于单细胞测序实验。