Endocardial Brg1 disruption illustrates the EMT origins of semilunar valve disease

Discreet defects during prenatal semilunar valve (SLV) development frequently progress to pathological states later in life and often require valve replacement surgery. As such, it is challenging to distinguish between the disrupted developmental processes, and their genetic and environmental influences, that trigger valve defects from mechanisms that drive the progression of an anatomically abnormal valve into a disease state. This distinction, which is essential to inform the rationale design of diagnostics and therapeutics, requires carefully characterizing when and where an implicated gene or pathway functions during valve development and/or homeostasis. Disrupted growth, differentiation, and patterning events that trigger SLV disease are coordinated by gene expression changes in endocardial, myocardial, and cushion mesenchymal cells. We explored the roles of chromatin regulation in valve gene regulatory networks via conditional inactivation of the mouse Brg1 associated factor (BAF) chromatin-remodeling complex in the endocardial lineage. Endocardial Brg1-deficient embryos develop thickened and mal-patterned SLV cusps that frequently become bicuspid and myxomatous, including in surviving adults. These SLV disease-like phenotypes originate from deficient endocardial-mesenchymal transformation (EMT) in the proximal outflow tract (pOFT) cushions. Mesenchymal cells of neural crest or other cardiac origins subsequently replace the missing EMT-derived cells but are incompetent to pattern the valve interstitium into regions with distinct extracellular matrix composition. Transcriptomics reveal genes that may promote growth and patterning of SLVs and/or serve as biomarkers of their diseased state. Mechanistic studies of SLV disease genes will distinguish between disease origins and progression; the latter may largely reflect secondary responses to a disrupted developmental system. Examination of transcriptional changes (RNAseq) in E14.5 dissected cardiac cushions (two paired samples) of Nfatc1Cre;Brg1F/F mutants and wildtype littermates产前半月瓣（semilunar valve, SLV）发育过程中出现的隐匿性缺陷，常于日后进展为病理状态，往往需要接受瓣膜置换手术。因此，要区分引发瓣膜缺陷的发育过程紊乱及其遗传与环境影响因素，与驱动解剖结构异常瓣膜进展为疾病状态的机制，极具挑战性。这一区分对于合理设计诊断与治疗方案至关重要，需要精准刻画相关基因或通路在瓣膜发育及/或稳态维持过程中的时空功能。引发SLV疾病的生长、分化及模式形成紊乱，由心内膜细胞、心肌细胞及心垫间质细胞的基因表达变化协同调控。本研究通过在心内膜谱系中条件性敲除小鼠Brg1相关因子（Brg1 associated factor, BAF）染色质重塑复合物，探究了染色质调控在瓣膜基因调控网络中的作用。心内膜Brg1缺陷的胚胎会出现SLV瓣叶增厚、形态异常，常进展为二叶瓣及黏液瘤样病变，存活至成年的个体亦可见此类表型。这类SLV疾病样表型源于近端流出道（proximal outflow tract, pOFT）心垫中的心内膜-间质转化（endocardial-mesenchymal transformation, EMT）功能缺陷。随后，神经嵴或其他心脏起源的间质细胞会填补缺失的EMT来源细胞，但无法将瓣膜间质重塑为具有特定细胞外基质组成的区域。转录组学分析揭示了一批可促进SLV生长与模式形成、或可作为其疾病状态生物标志物的基因。对SLV疾病相关基因的机制研究，可区分疾病的起源与进展：后者在很大程度上可反映发育系统紊乱引发的继发性反应。本研究对胚胎发育第14.5天（E14.5）解剖分离的Nfatc1Cre;Brg1F/F突变体及其野生型同窝仔鼠的心脏垫（2对配对样本）开展了转录组变化（RNA测序，RNAseq）分析。