scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3

Bowel function requires coordinated activity of many enteric neuron subtypes. Clear definition of subtype-specific gene expression may facilitate molecular diagnoses for bowel motility disorders. Using adult mouse colon RNAseq data from 635 myenteric neurons and 707 E17.5 neurons, we defined seven adult myenteric neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially-expressed genes. Manually dissected human colon myenteric plexus yielded data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells, and 2153 macrophages. Immunohistochemistry demonstrated differential protein abundance for BNC2, PBX3, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ~50% of myenteric neurons with distinct effects on smooth muscle contractions. This insight into enteric nervous system biology provides a foundation for future studies of bowel motility disorders. 20 scRNAseq datasets were analyzed: (1) 3 single-nucleus seq runs from mouse distal colon, (2) 16 single nucleus seq samples from human, (3) 1 single cell seq from E17.5 mouse whole bowel肠道功能依赖于多种肠神经元亚型的协同活动。明确亚型特异性基因表达特征，有助于肠道动力障碍的分子诊断。本研究利用来自635个肌间神经元与707个E17.5（胚胎17.5天）小鼠神经元的成年小鼠结肠RNA测序（RNAseq）数据，鉴定出7种成年肌间神经元亚型、8种E17.5神经元亚型及数百个差异表达基因。通过手动解剖获取的人结肠肌间神经丛样本，涵盖了48个神经元、3798个神经胶质细胞、5568个平滑肌细胞、377个间质细胞与2153个巨噬细胞的测序数据。免疫组织化学实验证实，BNC2、PBX3、RBFOX1、TBX2与TBX3在不同肠神经元亚型中存在蛋白丰度差异。条件性敲除Tbx3可减少表达NOS1的肌间神经元数量。结合Gfra1与Gfra2的差异表达特征与钙成像实验结果，本研究发现胶质细胞源性神经营养因子（GDNF）与神经营养因子（neurturin）可对约50%的肌间神经元活动产生急性差异化调控，并对平滑肌收缩产生截然不同的影响。本研究对肠神经系统生物学的深入解析，为后续肠道动力障碍相关研究奠定了坚实基础。本研究共分析了20个单细胞RNA测序（scRNAseq）数据集：(1) 3组小鼠远端结肠单细胞核测序结果；(2) 16组人类样本单细胞核测序样本；(3) 1组E17.5小鼠全肠单细胞测序结果。