Single Cell RNA-sequencing analysis of the juvenile and embryonic mouse enteric nervous system (ENS) of the small intestine

The study aimed to make a molecular definition of myenteric neuron classes in the mouse small intestine and describe their differentiation from enteric stem cells during embryogenesis. Method: We performed single cell RNA-sequencing of enteric neurons from small intestine at the juvenile stage (postnatal day (P)21) and ENS cells from the small intestine at embryonic day (E)15.5 and E18.5. We confirmed novel marker genes for all classes using immunohistochemistry and characterised the morphology/axons of three of newly discovered ENS neuron types. Results: 12 unique enteric neuron classes (ENC1-12) were identified and confirmed in native tissue by histochemical methods. The communication machinery (ligand, receptors, adhesion molecules), axonal patterns and morphologies were presented to infer functional roles of the ENCs. Analysis of embryonic transcriptomes revealed that enteric stem cells differentiate into two neuronal branches, that successively diversify into ENC1-7 and ENC8-12, respectively. Conclusion: Utilizing single cell RNA-sequencing we conclude that murine small intestine consists of 12 major myenteric classes that emerge through a step-wise fashion during embryonic development. Reference: Morarach, Mikhailova et al., 2020. BioRxiv 2020.03.02.955757 We isolated single myenteric neurons from pan-neuronal Baf53bCre;R26R-Tomato mice (P21) and single ENS cells (including enteric stem cells, glia and neurons) from Wnt1-Cre;R26R-Tomato embryos (E15.5 and E18.5) using flow cytometry and obtained transcriptomes by single cell RNA-sequencing (10xGenomics).本研究旨在对小鼠小肠肌间神经元类群开展分子定义，并阐明其在胚胎发育过程中由肠干细胞分化的动态过程。方法：我们针对幼年阶段（出生后第21天，P21）小鼠小肠的肠神经元，以及胚胎第15.5天（E15.5）与第18.5天（E18.5）的小鼠小肠肠神经系统（Enteric Nervous System, ENS）细胞开展了单细胞RNA测序（single cell RNA-sequencing）。通过免疫组织化学实验，我们验证了所有神经元类群的新型标记基因，并对三种新发现的肠神经系统神经元类型的形态与轴突特征进行了系统表征。本研究借助流式细胞术，从泛神经元特异性标记的Baf53bCre;R26R-Tomato幼年小鼠（P21）中分离单个肌间神经元，同时从Wnt1-Cre;R26R-Tomato胚胎小鼠（E15.5和E18.5）中分离得到包含肠干细胞、胶质细胞与神经元在内的单个肠神经系统细胞，最终通过10xGenomics平台完成单细胞RNA测序以获取转录组数据。结果：本研究通过组织化学方法在原生组织中鉴定并验证了12种独特的肠神经元类群（Enteric Neuron Classes, ENC1-12）。我们对各类肠神经元的信号传递机制（配体、受体、黏附分子）、轴突模式与形态特征进行了分析，以此推断其潜在的功能角色。对胚胎转录组的分析显示，肠干细胞可分化为两条神经元分化分支，二者随后分别进一步分化为ENC1-7与ENC8-12类群。结论：本研究借助单细胞RNA测序技术得出结论：小鼠小肠中共存在12种主要的肌间神经元类群，这些类群在胚胎发育过程中通过逐步分化的方式依次产生。参考文献：Morarach、Mikhailova等，2020。BioRxiv 2020.03.02.955757。