Sequential Notch activation regulates ventricular chamber development. Mus musculus

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. Overall design: 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

心室腔是支撑生命全程中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会引发严重的人类心脏缺陷。目前，心肌纤维早期形成的肌小梁网（trabecular meshwork）如何发育为成熟心室腔，其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）以维持肌小梁形成（trabeculation），后续则由糖基转移酶（glycosyltransferase）Manic Fringe（Manic Fringe, Mfng）介导的配体信号时序调控，协调心室模式构建与压实过程，并联合冠状血管发育共同促成成熟心室腔的形成。心内膜早期表达的Mfng会偏好激活Dll4-Notch1信号通路，进而诱导发育中心室的肌小梁形成。而心室的成熟与压实则需要心内膜中Mfng与Dll4的表达下调，这一过程会使心肌层的Jag1与Jag2信号能够激活该组织中的Notch1受体。该信号平衡的时空紊乱会严重干扰心脏心室腔的发育形成。本研究结果为心肌病的发病机制研究开辟了全新方向。

整体实验设计：
采用Nfact1或/和Tie2驱动的Cre重组酶（Cre）介导的Dll4与Notch1条件性敲除（conditional knockout, KO）模型：每个生物学重复的RNA提取自8个（Nfact1组）或9个（Tie2组）E9.5期胚胎的混合全心脏。实验分组包括：Dll4flox;Nfatc1-Cre敲除组与野生型（wild type, WT）同窝对照组（4个敲除重复与4个野生型重复）、Notch1flox;Nfatc1-Cre敲除组与野生型同窝对照组（3个敲除重复与2个野生型重复）、Dll4flox;Tie2-Cre敲除组与野生型同窝对照组（3个敲除重复与3个野生型重复）。
采用cTnT驱动的Cre重组酶介导的Jag1、Jag2及Jag1Jag2联合条件性敲除模型：每个生物学重复的RNA提取自4个E15.5期胚胎的混合心室。实验分组包括：Jag1flox;cTnT-Cre敲除组与野生型同窝对照组（3个敲除重复与3个野生型重复）、Jag2flox;cTnT-Cre敲除组与野生型同窝对照组（3个敲除重复与2个野生型重复）、Jag1flox;jag2flox;cTnT-Cre联合敲除组与野生型同窝对照组（3个敲除重复与2个野生型重复）。
采用Tie2驱动的Cre重组酶介导的MFng功能获得（Gain Of Function, GOF）模型：每个生物学重复的RNA提取自4个E15.5期胚胎的混合心室。实验分组包括：MFng;Tie2-Cre过表达组与野生型同窝对照组（4个过表达重复与4个野生型重复）。

测序文库制备与测序：针对Dll4、Notch1与Jag1敲除样本，采用标准Illumina TrueSeq RNA测序（RNA Seq）文库制备试剂盒构建文库，并使用Illumina GAIIx测序仪以75bp单端测序策略进行测序。针对Jag2、Jag1Jag2联合敲除样本及MFng功能获得样本，采用NEBNext Ultra RNA文库制备试剂盒（适配Illumina平台）构建文库，并使用Illumina HiSeq2500测序仪以61bp单端测序策略进行测序。