Molecular profiling of enteric nervous system cell lineages
Molecular profiling of enteric nervous system cell lineages
复制标题
DOI：
10.1038/s41596-022-00697-4
复制
发表时间：
2022-06-08
期刊：
影响因子：
14.8
通讯作者：
Pachnis，Vassilis
中科院分区：
文献类型：
--
作者：
Obata，Yuuki;Castano，Alvaro;Pachnis，Vassilis
肠神经系统（ENS）是肠神经元和神经胶质细胞的广泛网络，其是肠壁固有的并且调节肠生理学的几乎所有方面。虽然在理解调控ENS发育的遗传程序方面取得了相当大的进展，但对控制成体阶段ENS功能的分子途径的理解有限。推进成人ENS分子表征的限制之一涉及从成人肠组织中纯化健康神经元和胶质细胞的技术困难。为了克服这一点，我们开发了新的方法进行肠神经元和神经胶质细胞的转录组学分析，这是基于荧光标记的细胞核的分离。在这里，我们提供了一个一步一步的协议，成年小鼠肠神经元细胞核的标记使用腺相关病毒介导的基因转移，标记的细胞核的分离荧光分析，RNA纯化和核RNA测序。该方案也适用于分离肠神经元和神经胶质细胞核的肌间神经丛制剂从成年斑马鱼肠。最后，我们描述了一种用于肌间神经节中RNA可视化和量化的方法：颗粒核信号的空间整合（SIGNS）。通过遵循该方案，需要约3d来产生RNA并创建用于核RNA测序的cDNA文库，并且需要4d来对ENS组织进行高分辨率RNA表达分析。
The enteric nervous system (ENS) is an extensive network of enteric neurons and glial cells that is intrinsic to the gut wall and regulates almost all aspects of intestinal physiology. While considerable advancement has been made in understanding the genetic programs regulating ENS development, there is limited understanding of the molecular pathways that control ENS function in adult stages. One of the limitations in advancing the molecular characterization of the adult ENS relates to technical difficulties in purifying healthy neurons and glia from adult intestinal tissues. To overcome this, we developed novel methods for performing transcriptomic analysis of enteric neurons and glia, which are based on the isolation of fluorescently labeled nuclei. Here we provide a step-by-step protocol for the labeling of adult mouse enteric neuronal nuclei using adeno-associated-virus-mediated gene transfer, isolation of the labeled nuclei by fluorimetric analysis, RNA purification and nuclear RNA sequencing. This protocol has also been adapted for the isolation of enteric neuron and glia nuclei from myenteric plexus preparations from adult zebrafish intestine. Finally, we describe a method for visualization and quantification of RNA in myenteric ganglia: Spatial Integration of Granular Nuclear Signals (SIGNS). By following this protocol, it takes ~3 d to generate RNA and create cDNA libraries for nuclear RNA sequencing and 4 d to carry out high-resolution RNA expression analysis on ENS tissues.