Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypesin situ

Abstract

<jats:title>A<jats:sc>bstract</jats:sc></jats:title><jats:sec><jats:title>BACKGROUND &amp; AIMS</jats:title><jats:p>The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (EN). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions.</jats:p></jats:sec><jats:sec><jats:title>METHODS</jats:title><jats:p>Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA-Seq. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA-Seq (snRNA-Seq), totaling over four billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse snRNA-Seq data. Comparative informatics between human and mouse datasets identified shared EN subtype markers, which were visualized <jats:italic>in situ</jats:italic> using hybridization chain reaction (HCR).</jats:p></jats:sec><jats:sec><jats:title>RESULTS</jats:title><jats:p>Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of one or more marker genes.</jats:p></jats:sec><jats:sec><jats:title>CONCLUSIONS</jats:title><jats:p>Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human GI studies.</jats:p><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:caption><jats:title>Graphical Abstract</jats:title></jats:caption><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="187211v1_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec>

Regional cytoarchitecture of the adult and developing mouse enteric nervous system

“…30,31 Our analysis of enteric neuron subtypes throughout the intestines reveals striking differences in distribution for many neurotransmitters, neuropeptides, and CBPs across intestinal regions and neuronal stripes. Regional differences in neuronal marker distribution have been observed previously, 9,32 and such differences are perhaps unsurprising, considering the specific functions of the SI, colon, and subregions thereof, and regional differences in extrinsic ENS innervation. [33][34][35][36] Despite this, few studies have specifically investigated regionality, with many focusing on the ileum 37 or pooling entire organs.…”

Neonatal antibiotics have long term sex‐dependent effects on the enteric nervous system

“…But there was significant disruption in the myenteric plexus of young adult males with reduced numbers of neurons (and glia) and altered proportions of several subtypes of putative cholinergic neurons, but not nitrergic neurons. Recent single neuron transcriptomic studies (Drokhlyansky et al., 2020; May‐Zhang et al., 2021; Morarach et al., 2021; Wright et al., 2021) and other studies using combined reporter and immunohistochemical analyses (Nestor‐Kalinoski et al., 2022) indicate that neurons can only be subtyped into presumed functional groups by combinations of several key markers. Nevertheless, significant conclusions about the functional identities of the neurons disrupted by neonatal antibiotics can be drawn from our study.…”

A contamination focused approach for optimizing the single-cell RNA-seq experiment

“…Nuclei also have smaller RNA pools that result in lower capture efficiencies and biological signals, which can be overwhelmed by ambient RNA. While snRNA-seq makes single-cell analysis possible for frozen and difficult-to-dissociate tissues (May-Zhang et al, 2021), end users should balance these factors when deciding whether to use cells or nuclei for their single-cell analysis.…”