An image-based transcriptomic atlas of the mouse gut reveals spatial, regional, and microbiota-dependent fine-tuning

The gastrointestinal tract (GI) is a remarkably complex environment comprising a diverse array of cell types, microbes, and bioactive small molecules. To shed new light on how the gut is organized and senses its environment, we constructed a spatially resolved single-cell atlas of the mouse lower digestive tract using MERFISH with a specific focus on the thousands of receptors involved in small molecule sensation and microbial pattern recognition. We identified expected and novel cell types and charted their spatial organization across four GI regions. In addition, we discovered receptor expression gradients and heterogeneity cued by spatial location, and overall revealed divergent sensing capabilities across gut cell types, regions, and spatial contexts. In addition, we charted the remodeling of these features that occurs in the absence of the microbiome. Our atlas opens a new window into the molecular and cellular organization of gut sensation, which, given the pharmacological relevance of many receptors, may provide a clinically relevant resource. To provide an in-house measurement of gene expression in the SPF intestine as a validation for MERFISH results, distal colon tissue was harvested from C57BL/6NTac mice, flushed and rinsed with 4 mM ribonucleoside vanadyl complex (RVC; New England Biolabs [NEB] S1402S) in 1 x phosphate-buffered saline (PBS; ThermoFisher Scientific[Thermo] AM9625) at 4 oC. The tissue was then dissolved in TRIzol™ (Thermo 15596026) and RNA extraction was performed by the Direct-zol RNA Miniprep Plus kit (Zymo R2071), followed by low input library preparation and paired-end sequencing by Illumina NextSeq 500. To analyze these data, we used tophat (v2.1.1) and cufflinks (v2.2.1) with default parameters, aligning to the mouse genome (GRCm39).