Study: Genetic encoding of an esophageal motor circuit 288 cells

This dataset is from a single-nuclei RNA-seq (sNuc-seq) analysis of cholinergic neuron nuclei in and around the mouse nucleus ambiguus (nAmb). A summary of the study and methods are provided below.

STUDY SUMMARY Motor control of the striated esophagus originates in the nucleus ambiguus (nAmb), a vagal motor nucleus which also contains upper airway motor neurons and parasympathetic preganglionic neurons for the heart and lungs. We disambiguate nAmb neurons based on their genome-wide expression profiles, efferent circuitry, and ability to control esophageal muscles. Our single-cell RNA-sequencing analysis predicts three molecularly distinct nAmb neuron subtypes and annotates them by subtype-specific marker genes: Crhr2, Vipr2, and Adcyap1. Mapping the axon projections of the nAmb neuron subtypes reveals that Crhr2nAmb neurons innervate the esophagus, raising the possibility that they control esophageal muscle function. Accordingly, focal optogenetic stimulation of cholinergic Crhr2+ fibers in the esophagus results in contractions. Activating Crhr2nAmb neurons has no effect on heart rate, a key parasympathetic function of the nAmb, whereas activating all nAmb neurons robustly suppresses heart rate. Together these results reveal a genetically defined circuit for motor control of the esophagus.

METHODS The ventrolateral medulla of five male Chat-Cre mice were injected with a Cre-dependent reporter virus, AAV-DIO-H2b-mCherry. Four weeks later, at the age of 27 weeks, mice were rapidly decapitated for brain extraction to avoid stress related changes in nuclear mRNA. Following immediate brain extraction, 1 mm thick coronal sections of hindbrain through the nAmb’s full rostral-caudal extent (Bregma -6.5 mm to -8.0 mm) were cut and immersed in ice-cold RNA-later (Qiagen catalog # 76106). After at least 30 minutes in ice-cold RNA-later, the nAmb was visualized under a fluorescence stereomicroscope (Zeiss Discovery V8) and dissected, then stored in RNA-later overnight at 4°C. On the next day, nAmb tissue was homogenized and purified by density-gradient centrifugation into a single-nuclei suspension as previously described (Habib et al., 2016; Todd et al., 2020). The single-nuclei suspension was sorted by FACS to isolate one H2b-mCherry+ nucleus per well of three 96-well plates, which were then centrifuged at 2,500rcf, frozen on dry ice, and stored at -80°C until use. After purifying the RNA by RNA-clean SPRI reagents (1.5x ratio of SPRI to sample), cDNA libraries were generated from polyadenylated mRNA of each sample using Smart-Seq2 (Picelli et al., 2014). Illumina sequencing libraries were made from single-nuclei cDNA samples as described previously (Tao et al., 2021) and then sequenced by Illumina Next-Seq 500. Reads were demultiplexed by bcl2fastq2 v2.20.0 (Illumina) and aligned to the mouse genome by STAR v2.6.1 (Dobin et al., 2013). Duplicates were removed with Picard Tools v2.18.21. Aligned reads were processed into a digital gene expression (DGE) file with Drop-Seq Tools v2.3.0 and tagged using ‘‘GENCODE_M16_PRI’’ annotation. Since the sNuc-seq protocol does not produce true UMIs, we considered any unique read (i.e., non-duplicated) as a unique “UMI-tagged” read for the Drop-Seq pipeline. An R software package for single-cell genomics analysis, Seurat v4.0 (Hao et al., 2021), was used to filter, scale, and normalize the data, then perform dimensionality reduction with principal component (PC) analysis on the top 2,000 most variable genes, cell clustering on the top 6 PCs, and differential expression analysis using Wilcoxon Rank Sum test and default settings, as previously described (Tao et al., 2021; Todd et al., 2020).