Gut Online First, published on February 26, 2018 as 10.1136/gutjnl-2017-315631 Neurogastroenterology ORIGINAL ARTICLE Gut: first published as 10.1136/gutjnl-2017-315631 on 26 February 2018. Downloaded from Single-cell RNAseq reveals seven classes of colonic sensory neuron James R F Hockley,1,2 Toni S Taylor,1 Gerard Callejo,1 Anna L Wilbrey,2 Alex Gutteridge,2 Karsten Bach,1 Wendy J Winchester,2 David C Bulmer,1 Gordon McMurray,2 Ewan St John Smith1 ► Additional material is ABSTRact pathways to the central nervous system (CNS).1 In published online only. To view Objective Integration of nutritional, microbial and the colorectum, sensory innervation is organised please visit the journal online (http:// dx. doi. org/ 10. 1136/ inflammatory events along the gut-brain axis can alter into two main pathways: thoracolumbar (TL) spinal gutjnl- 2017- 315631). bowel physiology and organism behaviour. Colonic afferents projecting via the lumbar splanchnic sensory neurons activate reflex pathways and give nerve (LSN) and lumbosacral (LS) spinal afferents 1Department of Pharmacology, University of Cambridge, rise to conscious sensation, but the diversity and projecting via the pelvic nerve (PN) that are respon- Cambridge, UK division of function within these neurons is poorly sible for transducing conscious sensations of full- 2Neuroscience and Pain understood. The identification of signalling pathways ness, discomfort, urgency and pain, in addition to Research Unit, Pfizer, contributing to visceral sensation is constrained by a reflex actions.2 Cambridge, UK paucity of molecular markers. Here we address this by Visceral sensory afferents act to maintain many comprehensive transcriptomic profiling and unsupervised aspects of GI physiology, such as continence and Correspondence to James R F Hockley, Department clustering of individual mouse colonic sensory neurons. evacuatory function and contribute to local regu- of Pharmacology, University Design Unbiased single-cell RNA-sequencing was lation of motility, secretory processes and blood of Cambridge, Cambridge, performed on retrogradely traced mouse colonic flow.2 Locally, peripheral visceral afferent fibres UK; jh2033@ cam. ac. uk sensory neurons isolated from both thoracolumbar (TL) interact with enteroendocrine cells in the mucosa and Dr Ewan St John Smith, and lumbosacral (LS) dorsal root ganglia associated Department of Pharmacology, and immune cells in the gut wall to initiate and 3 University of Cambridge, with lumbar splanchnic and pelvic spinal pathways, modulate sensory input. Sensory pathways are a Cambridge CB1 2PD, UK; respectively. Identified neuronal subtypes were alidatedv major interface between luminal contents, including es336@ cam. ac. uk by single-cell qRT-PCR, immunohistochemistry (IHC) and gut microbiota, the immune system and the CNS, Ca2+-imaging. Received 12 November 2017 providing the capacity to modulate mood and sensa- 4 Revised 2 February 2018 Results Transcriptomic profiling and unsupervised tion. The importance of a healthy visceral sensory http://gut.bmj.com/ Accepted 10 February 2018 clustering of 314 colonic sensory neurons revealed nervous system is demonstrated when these sensory seven neuronal subtypes. Of these, five neuronal pathways are dysregulated or maladapted, such as subtypes accounted for 99% of TL neurons, with LS following spinal cord injury and in inflammatory neurons almost exclusively populating the remaining bowel disease, where loss of effective bowel func- two subtypes. We identify and classify neurons based tion and long-term chronic pain leads to significant on novel subtype-specific marker genes using single- morbidity.5 6 Indeed, in patients with IBS, chronic cell qRT-PCR and IHC to validate subtypes derived from 2+ dysregulation of afferent signalling is thought to on September 30, 2021 by guest. Protected copyright. RNA-sequencing. Lastly, functional Ca -imaging was drive visceral hypersensitivity.7 conducted on colonic sensory neurons to demonstrate Significant efforts have been taken to characterise subtype-selective differential agonist activation. the diversity of visceral afferent fibres innervating the Conclusions We identify seven subtypes of colonic colon in order to fully understand their function in both sensory neurons using unbiased single-cell RNA- normal GI physiology and disease. Visceral afferents sequencing and confirm translation of patterning have been classified by morphological characteristics to protein expression, describing sensory diversity (eg, peripheral ending structure and location within encompassing all modalities of colonic neuronal the gut wall), by expression of ion channels, recep- sensitivity. These results provide a pathway to molecular tors and neurotransmitters and by functional criteria interrogation of colonic sensory innervation in health and (eg, basal firing rate, mechanical activation threshold disease, together with identifying novel targets for drug and stimulus-response functions). Consequently, a development. diverse range of visceral nerve classifications exist, which vary according to the particular pathway and species investigated, as well as the investigator and INTRODUCTION experimental protocols used. While recent schema The gastrointestinal (GI) tract is a complex set of to integrate this myriad of classifications proposes organs responsible for the ingestion and digestion the presence of five major structural types of endings To cite: Hockley JRF, of food, absorption of nutrients and evacuation of (mucosal, intraganglionic laminar, muscular-mucosal, Taylor TS, Callejo G, et al. Gut 2 Epub ahead of print: [please waste. In addition to autonomic pathways, the GI intramuscular and vascular afferents ), traditional include Day Month Year]. tract can evoke behavioural changes by providing molecular criteria describe a relatively homogeneous doi:10.1136/ conscious awareness of damage, such as during population of peptidergic,8 voltage-gated sodium gutjnl-2017-315631 injury, inflammation and infection, through sensory channel 1.8-expressing ‘nociceptors’.9 10 Hockley JRF, et al. Gut 2018;0:1–12. doi:10.1136/gutjnl-2017-315631 1 Copyright Article author (or their employer) 2018. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Neurogastroenterology Isolation of mouse colonic sensory neurons Significance of this study TL and LS dorsal root ganglia (DRG) were collected from healthy Gut: first published as 10.1136/gutjnl-2017-315631 on 26 February 2018. Downloaded from mice 3–10 days after retrograde tracing from the colon by Fast What is already known on this subject? Blue (FB) injection using previously described methods.11 12 Indi- ► Sensory dysfunction is implicated in the pathogenesis of GI vidual colonic sensory neurons were manually picked by pulled disease, and the modulation of sensory pathways are an glass pipette on an adapted inverted microscope after enzymatic important site of action for existing pharmacotherapies. dissociation of the DRG and were used in subsequent single-cell ► Colonic sensory neurons that control reflex pathways and RNAseq (scRNAseq) and qRT-PCR analysis. Prior to picking, give rise to sensations of urge, discomfort and pain are cells were photographed for cell size analysis.10 For immuno- currently classified by differences in their sensitivity to histochemistry (IHC), FB-labelled mice were perfused-fixed and mechanical stimuli and anatomical structure of their nerve whole TL and LS DRG processed. endings. ► Existing schema propose five major subpopulations of colorectal afferents, while traditional molecular criteria Single-cell RNAseq and clustering describe a relatively homogenous population of peptidergic, Full-length cDNA from polyadenylated RNA of 399 colonic 13 sensory neurons were generated using Smart-seq2 protocols voltage-gated sodium channel NaV1.8-expressing neurons; however, these do not fully encapsulate all sensory and multiplexed into six pooled libraries using Illumina Nextera modalities present in the gut. XT DNA Sample Kit before 75 bp paired-end sequencing on an Illumina NextSeq500 (read depth ~4.9M reads/cell). What are the new findings? Sample-specific reads were aligned to the mouse reference ► Using single-cell RNA sequencing of mouse colonic sensory genome (GRCm38.p3; Ensembl V.80) and genomic features neurons, we describe a comprehensive framework for the determined using featureCounts. Low-quality cells were filtered molecular basis of visceral sensation from the colorectum. (online supplementary figure 1) resulting in normalised data from ► We identify seven distinct colonic sensory neuron subtypes 325 cells and 34 769 genes14 15 being passed onto downstream with nerve-specific subtype diversity between the lumbar clustering analyses. Latent technical effects14 and contaminating splanchnic nerve and pelvic nerve. satellite glia were conservatively removed16 (online supplemen- ► We characterise a range of novel molecular markers for tary figure 2) before clustering the remaining 314 cells based mouse colonic neuron subtypes facilitating interrogation of on gene expression profiles using single-cell consensus clustering these pathways to GI function in health and disease. (SC3)17 achieving greatest stability with k=7. Cluster robustness was investigated using bootstrapping of a downsampled dataset How might it impact on clinical practice in the foreseeable (online supplementary figure 3). Using SC3, we identified 709 future? marker genes capable of distinguishing individual clusters (area ► Molecular fingerprinting