Gpr126 is essential for placental development

Gpr126 is an adhesion G protein-coupled receptor (GPCR) that is expressed in the endocardium of the heart and its inactivation in mice leads to embryonic lethality and defective ventricular trabeculation. However, the mechanistic bases of this phenotype are unknown. We generated a series of standard, conditional loss- and gain-of-function alleles in mice and zebrafish, and found that cardiac abnormalities associated with global Gpr126 inactivation are secondary defects that arise as a result of placental insufficiency. Total RNA was extracted from E12.5 mouse embryonic hearts after removing atria and the valve region in sterile ice-cold PBS. 4 pools of Gpr126+/+ (WT) and 4 pools of Gpr126delta7/delta7 (KO) embryonic ventricles (4 ventricles per pool) were used. The tissue was homogenized in TRI ReagentTM Solution (Invitrogen, AM9738) using steel beads in the TissueLyser LT Adapter for 5 minutes at 50 Hz. For RNA extraction, the PicoPureTM RNA Isolation kit (Applied Biosystems, KIT0204) was used according to the manufacturer's instructions. RNA was quantified and its purity checked with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA integrity was verified with an Agilent 2100 Bioanalyzer instrument (Agilent Technologies).Gpr126是一种黏附型G蛋白偶联受体（adhesion G protein-coupled receptor, GPCR），在心脏心内膜中表达；该基因在小鼠体内失活会导致胚胎致死与心室肌小梁发育异常，但其对应的分子机制目前尚不明确。我们在小鼠与斑马鱼中构建了一系列标准化的条件性功能丧失与功能获得等位基因，研究发现全身性Gpr126失活所引发的心脏异常实为胎盘功能不全导致的继发性缺陷。我们采用无菌冰浴磷酸盐缓冲液（PBS）去除心房与瓣膜区域后，从胚胎发育第12.5天（E12.5）的小鼠胚胎心脏中提取总RNA。实验共设置4组野生型（wild type, WT）胚胎心室混合样本与4组Gpr126delta7/delta7基因敲除（knockout, KO）胚胎心室混合样本，每组混合样本包含4个胚胎心室。将组织置于TRI Reagent™溶液（Invitrogen，货号AM9738）中，使用钢珠在TissueLyser LT适配器中以50Hz频率均质处理5分钟。采用PicoPure™ RNA提取试剂盒（Applied Biosystems，货号KIT0204），按照试剂盒说明书进行RNA提取操作。使用NanoDrop ND-1000分光光度计（Thermo Scientific）对RNA进行定量与纯度检测。采用Agilent 2100生物分析仪（Agilent Technologies）验证RNA完整性。