Sequential Notch activation regulates ventricular chamber development

Ventricular chambers are essential for the rhythmic contraction and relaxation that occurs in every hearbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is still poorly understood. Here we show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation and later coordinates ventricular patterning and compaction with coronary vessel development to give rise to the mature chamber via a temporal sequence of ligand signalling determined by the glycosyltransferase Manic Fringe (Mfng). The early endocardial expression of Mfng favours Dll4-Notch1 signalling, Which induces trabeculation in the developing ventricle.Ventricular maturation and compaction in turn require Mfng and Dll4 downregulation in the endocardium, Which allows myocardial Jag1- And Jag2- Signalling to Notch1 in this tissue.Timely and spatial perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies. Dll4 and Notch1 conditional KOs using Nfact1 and/or Tie2 driven Cre expression: RNA was isolated from pooled whole hearts of 8 (Nfact1) or 9 (Tie2) E9.5 embryos per replicate. Dll4flox;Nfatc1-Cre and WT siblings (4 KO and 4 WT replicates), Notch1flox;Nfatc1-Cre and WT siblings (3 KO and 2 WT replicates), Dll4flox;Tie2-Cre and WT siblings (3 KO and 3 WT replicates). Jag1, Jag2 and Jag1Jag2 conditional KOs using cTnT driven Cre expression: RNA was isolated from pooled heart ventricles of 4 E15.5 embryos per replicate. Jag1flox;cTnT-Cre and WT siblings (3 KO and 3 WT replicates), Jag2flox;cTnT-Cre and WT siblings (3 KO and 2 WT replicates). Jag1flox;jag2flox;cTnT-Cre and WT siblings (3 KO and 2 WT replicates). MFng Gain Of Function using Tie2 driven Cre expression: RNA was isolated from pooled heart ventricles of 4 E15.5 embryos per replicate. MFng;Tie2-Cre and WT siblings (4 GOF and 4 WT replicates). For Dll4, Noth1 and Jag1 KOs, libraries were prepared using the standard Illumina TrueSeq RNASeq library preparation kit and sequenced in a GAIIx Illumina sequencer using a 75bp single end elongation protocol. For Jag2 and Jag1Jag2 KOs and MFng GOF libraries were prepared prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina and sequenced in a HiSeq2500 Illumina sequencer using a 61bp single end elongation protocol心室腔（ventricular chambers）是维持终生每次心跳节律性收缩与舒张的核心结构。心室腔发育过程中的先天性异常（congenital abnormalities）会引发严重的人类心脏缺陷。目前，由心肌纤维构成的早期小梁网（trabecular meshwork）的形成过程，及其后续向成熟心室腔的发育机制仍未被充分阐明。本研究发现，Notch信号通路（Notch signalling）首先通过连接心室腔心内膜（endocardium）与心肌层（myocardium）以维持小梁形成（trabeculation）；随后，由糖基转移酶Manic Fringe（Mfng）调控配体信号的时序序列，协同心室模式构建（ventricular patterning）、致密化（compaction）过程与冠状血管发育（coronary vessel development），最终促成成熟心室腔的形成。Mfng在早期心内膜的表达倾向于激活Dll4-Notch1信号通路，进而诱导发育中心室的小梁形成。而心室成熟与致密化过程，则需要心内膜中Mfng与Dll4的表达下调，这一变化使得心肌层的Jag1与Jag2信号能够激活该组织中的Notch1。该信号平衡的时序与空间扰动，会严重干扰心脏心室腔的发育。本研究结果为心肌病的发病机制研究开辟了全新的方向。 采用Nfact1或Tie2驱动的Cre重组酶（Cre expression）体系的Dll4与Notch1条件性基因敲除（conditional knockout）实验：每个重复样本取自8个（Nfact1组）或9个（Tie2组）E9.5胚胎的混合全心脏RNA。具体分组包括：Dll4flox;Nfatc1-Cre组与野生型同窝对照组（WT siblings，各4个敲除组重复样本与4个野生组重复样本），Notch1flox;Nfatc1-Cre组与野生型同窝对照组（3个敲除组重复样本与2个野生组重复样本），Dll4flox;Tie2-Cre组与野生型同窝对照组（各3个敲除组重复样本与3个野生组重复样本）。 采用cTnT驱动的Cre重组酶体系的Jag1、Jag2及Jag1Jag2双条件性敲除实验：每个重复样本取自4个E15.5胚胎的混合心室RNA。具体分组包括：Jag1flox;cTnT-Cre组与野生型同窝对照组（3个敲除组重复样本与3个野生组重复样本），Jag2flox;cTnT-Cre组与野生型同窝对照组（3个敲除组重复样本与2个野生组重复样本），Jag1flox;jag2flox;cTnT-Cre组与野生型同窝对照组（3个敲除组重复样本与2个野生组重复样本）。 采用Tie2驱动的Cre重组酶体系的Mfng功能获得性突变（gain of function，GOF）实验：每个重复样本取自4个E15.5胚胎的混合心室RNA；MFng;Tie2-Cre组与野生型同窝对照组（4个功能获得组重复样本与4个野生组重复样本）。 针对Dll4、Notch1及Jag1的敲除实验，采用标准Illumina TrueSeq RNA测序文库制备试剂盒（Illumina TrueSeq RNASeq library preparation kit）构建文库，并使用Illumina GAIIx测序仪（Illumina GAIIx sequencer）以75bp单端延伸测序（single end elongation protocol）策略进行测序。针对Jag2、Jag1Jag2双敲除及Mfng功能获得性突变实验的文库，则采用NEBNext Ultra RNA文库制备试剂盒（NEBNext Ultra RNA Library Prep Kit for Illumina）构建，并使用Illumina HiSeq2500测序仪（Illumina HiSeq2500 sequencer）以61bp单端延伸测序策略进行测序。