ENSMAP: Molecular and Functional Mapping of the Enteric Nervous System

项目总结 胃肠(GI)运动和排便是营养吸收、粪便排出和整体健康的绝对先决条件。正常的胃肠动力、血管灌流和肠道炎症由位于肠壁固有的肠神经系统(ENS)神经节内的大量神经元协调。虽然最近的工作已经确定了指导前体细胞初始发育的各种基因，这些前体细胞可以在肠壁形成肠神经元，但我们对成年肠神经元中表达的基因知之甚少。因此，我们无法确定产生肠道神经元的努力是否会产生正常的细胞类型补充。此外，我们并不完全了解不同类型的神经元如何对肠道运动的整体协调做出贡献，因为仅使用共同的免疫组织化学标记物并不能区分功能上不同的亚型。因此，我们瞄准并从功能上操纵肠道中特定类型神经元的能力极其有限。为了克服这些限制，我们的应用程序建议开发一个全面的、单细胞的正常小鼠肠神经细胞转录组图谱，同时对人类肠道不同区域的肠神经节进行深度测序，从而获得人类肠神经节的全球基因表达图谱。为了捕获单细胞RNA-Seq的小鼠肠道神经元，我们将使用我们开发的用于肠道神经元活细胞成像的荧光转基因小鼠品系。人肠神经节将通过激光捕获显微解剖从成人手术残留物中收集。对小鼠和人类数据集之间的肠道神经元表达谱进行比较，将确定标记不同神经元亚型的保守基因。由此得到的肠神经元表达图谱将确定离散神经元亚型的特定分子指纹，这些亚型对于在肠道不同区域进行有针对性的、功能性的胃肠动力操作至关重要。

PROJECT SUMMARY Gastrointestinal (GI) motility and defecation are absolute prerequisites for nutrient absorption, fecal elimination and overall health. Normal GI motility, vascular perfusion, and intestinal inflammation are coordinated by vast numbers of neurons that reside within ganglia of the enteric nervous system (ENS) intrinsic to the gut wall. While recent work has identified diverse genes that direct the initial development of progenitor cells that give rise to enteric neurons in the wall of the intestine, we know very little about the genes that are expressed in adult enteric neurons. Consequently we are unable to determine whether efforts to generate enteric neurons produce the normal complement of cell types. Moreover we do not fully understand how distinct types of neurons contribute to overall coordination of intestinal motility because the use of common immunohistochemical markers alone does not distinguish functionally distinct subtypes. As a result, our abilities to target and functionally manipulate specific types of neurons in the gut are extremely limited. To surpass these limitations, our application proposes to develop a comprehensive, single cell transcriptome map of adult enteric neurons in normal mice in parallel with deep sequencing of enteric ganglia from distinct regions of human intestine so that a global gene expression atlas of human enteric ganglia is obtained. To capture mouse enteric neurons for single cell RNA-Seq we will use a fluorescent transgenic mouse line that we developed for live-cell imaging of enteric neurons. Human enteric ganglia will be collected by laser capture microdissection from adult surgical remnants. Comparison of enteric neuron expression profiles between mouse and human data sets will identify conserved genes that mark distinct neuronal subtypes. The resulting expression atlas of enteric neurons will define specific molecular fingerprints for discrete neuron subtypes that are essential to pursue targeted, functional manipulation of GI motility in distinct regions of the intestine.