Department of Pediatrics, Abramson Research Center, Children's Hospital of Philadelphia Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Pittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, Pennsylvania; Center for Neuroscience at the University of Pittsburgh, Pittsburgh, Pennsylvania.
Cell Mol Gastroenterol Hepatol. 2021;11(5):1548-1592.e1. doi: 10.1016/j.jcmgh.2020.12.014. Epub 2021 Jan 11.
Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function.
To identify subtype-specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results.
RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, 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.
Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.
肠道功能需要不同肠神经元亚型的协调活动。我们的目的是确定这些神经元亚型的基因表达，以促进治疗破坏性肠神经病变的新疗法的发展，并更多地了解肠神经系统的功能。
为了鉴定亚型特异性基因，我们对成年小鼠和人类结肠肌间神经丛进行了单核 RNA-seq 分析，并对来自整个肠道的 E17.5 小鼠 ENS 细胞进行了单细胞 RNA-seq 分析。我们使用免疫组织化学、选择性突变小鼠和钙成像来验证和扩展结果。
对 635 个成年小鼠结肠肌间神经元和 707 个来自整个肠道的 E17.5 神经元进行 RNA-seq 鉴定了七个成年神经元亚型、八个 E17.5 神经元亚型和数百个差异表达基因。手动解剖的人结肠肌间神经丛从 48 个神经元、3798 个神经胶质细胞、5568 个平滑肌细胞、377 个 Cajal 间质细胞和 2153 个巨噬细胞中获得了 RNA-seq 数据。免疫组织化学显示 BNC2、PBX3、SATB1、RBFOX1、TBX2 和 TBX3 在肠神经元亚型中的表达存在差异。条件性 Tbx3 缺失减少了 NOS1 表达的肌间神经元。差异表达的 Gfra1 和 Gfra2 与钙成像相结合，揭示了 GDNF 和 NRTN 急性且差异地调节具有不同平滑肌收缩效应的约 50%肌间神经元的活性。
单细胞分析确定了肌间神经元亚型中差异表达的基因，以及 TBX3、GDNF 和 NRTN 的新作用。这些数据为肠道运动障碍的分子诊断研究和新疗法提供了便利。
