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Single-cell RNAseq reveals seven classes of colonic sensory neuron
Gut ( IF 24.5 ) Pub Date : 2018-02-26 , DOI: 10.1136/gutjnl-2017-315631
James R F Hockley , Toni S Taylor , Gerard Callejo , Anna L Wilbrey , Alex Gutteridge , Karsten Bach , Wendy J Winchester , David C Bulmer , Gordon McMurray , Ewan St John Smith

Objective Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons. Design Unbiased single-cell RNA-sequencing was performed on retrogradely traced mouse colonic sensory neurons isolated from both thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Identified neuronal subtypes were validated by single-cell qRT-PCR, immunohistochemistry (IHC) and Ca2+-imaging. Results Transcriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes. Of these, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We identify and classify neurons based on novel subtype-specific marker genes using single-cell qRT-PCR and IHC to validate subtypes derived from RNA-sequencing. Lastly, functional Ca2+-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation. Conclusions We identify seven subtypes of colonic sensory neurons using unbiased single-cell RNA-sequencing and confirm translation of patterning to protein expression, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity. These results provide a pathway to molecular interrogation of colonic sensory innervation in health and disease, together with identifying novel targets for drug development.

中文翻译:

单细胞 RNAseq 揭示了七类结肠感觉神经元

目的沿着肠-脑轴整合营养、微生物和炎症事件可以改变肠道生理和生物体行为。结肠感觉神经元激活反射通路并产生有意识的感觉,但对这些神经元内功能的多样性和划分知之甚少。导致内脏感觉的信号通路的鉴定受到缺乏分子标记的限制。在这里,我们通过综合转录组分析和单个小鼠结肠感觉神经元的无监督聚类来解决这个问题。设计 对分别从与腰内脏和骨盆脊髓通路相关的胸腰椎 (TL) 和腰骶 (LS) 背根神经节分离的逆行追踪小鼠结肠感觉神经元进行无偏单细胞 RNA 测序。通过单细胞 qRT-PCR、免疫组织化学 (IHC) 和 Ca2+ 成像验证确定的神经元亚型。结果 314 个结肠感觉神经元的转录组学分析和无监督聚类揭示了七种神经元亚型。其中,5 种神经元亚型占 TL 神经元的 99%,LS 神经元几乎完全占据其余两种亚型。我们使用单细胞 qRT-PCR 和 IHC 基于新的亚型特异性标记基因识别和分类神经元,以验证源自 RNA 测序的亚型。最后,对结肠感觉神经元进行功能性 Ca2+ 成像,以证明亚型选择性差异激动剂激活。结论我们使用无偏单细胞 RNA 测序鉴定了七种结肠感觉神经元亚型,并确认了模式到蛋白质表达的翻译,描述感官多样性,包括结肠神经元敏感性的所有形式。这些结果为健康和疾病中结肠感觉神经支配的分子研究提供了一条途径,同时也确定了药物开发的新靶点。
更新日期:2018-02-26
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