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scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3
Cellular and Molecular Gastroenterology and Hepatology ( IF 7.2 ) Pub Date : 2021-01-11 , DOI: 10.1016/j.jcmgh.2020.12.014
Christina M Wright 1 , Sabine Schneider 1 , Kristen M Smith-Edwards 2 , Fernanda Mafra 3 , Anita J L Leembruggen 4 , Michael V Gonzalez 3 , Deepika R Kothakapa 1 , Jessica B Anderson 1 , Beth A Maguire 1 , Tao Gao 1 , Tricia A Missall 5 , Marthe J Howard 6 , Joel C Bornstein 4 , Brian M Davis 2 , Robert O Heuckeroth 1
Affiliation  

Background and aims

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.

Methods

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.

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 RNAseq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells, 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.

Conclusion

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.



中文翻译:

scRNA 测序揭示了 GDNF、NRTN 和 TBX3 的新肠神经系统作用

背景和目标

肠功能需要不同肠神经元亚型的协调活动。我们的目标是定义这些神经元亚型中的基因表达,以促进开发治疗破坏性肠神经病的新型治疗方法,并了解更多关于肠神经系统功能的信息。

方法

为了识别亚型特异性基因,我们对成年小鼠和人结肠肌间神经丛进行了单核 RNA-seq,并对来自全肠的 E17.5 小鼠 ENS 细胞进行了单细胞 RNA-seq。我们使用免疫组织化学、选择突变小鼠和钙成像来验证和扩展结果。

结果

对 635 个成年小鼠结肠肌间神经元和来自全肠的 707 个 E17.5 神经元的 RNA-seq 定义了七种成年神经元亚型、八种 E17.5 神经元亚型和数百种差异表达基因。手动解剖人结肠肌间神经丛产生了来自 48 个神经元、3798 个胶质细胞、5568 个平滑肌、377 个间质细胞和 2153 个巨噬细胞的 RNAseq 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差异表达。条件性Tbx3损失减少了表达 NOS1 的肌间神经元。差分Gfra1Gfra2表达结合钙成像显示 GDNF 和 neurturin 敏锐地和差异地调节约 50% 的肌间神经元的活动,对平滑肌收缩有明显的影响。

结论

单细胞分析定义了在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。这些数据有助于肠动力障碍的分子诊断研究和新疗法。

更新日期:2021-01-11
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