Nat Neurosci: Based on single-cell RNA sequencing, it depicts the diversity of intestinal neuron species

The autonomous function of the gastrointestinal tract requires the joint activity of neurons with different functions of the gastrointestinal nervous system.
, the extent of intestinal neuron diversity and how it appears in the development process is still unknown.
to this end, the Marklund Ulrika team at the Karolinska School of Medicine in Sweden conducted research on intestinal neurons in mice using single-cell RNA sequencing techniques to understand the diversity of intestinal neuron species.
the latest findings, published online December 7 in the journal Nature Neuroscience.
and results of the study, the researchers concluded that the number of neurons previously reported was not sufficient to represent the diversity of neurons throughout the intestinal nervous system (ENS).
to enrich the proportion of neurons, the researchers used the pan-neuron Baf53b-Cre19 mice to mate with R26R-Tomato-reported mice, specific and effectively labeling neurons in the intestine.
, the co-expression of the TOM-reported gene of neuron marker HUC/D and intestinal glial cell marker SOX2/SOX10 was quantified in the muscle intestinal plexus in different areas of the small intestine.
10x RNA sequencing for single-cell analysis by using current cytomethry.
of the 9,141 cells captured, the researchers retained a total of 600 unique molecularly identified cell (UMI) counts to assess the proportion of neurons in the data set.
4,892 high-quality intestinal neurons were retained for unsealed graph-based clustering, resulting in 12 intestinal neuron categories (ENCs).
new classification identified four previously discovered clusters, redefined six clusters, and identified two new clusters.
researchers found that each of the 12 intestinal nervous systems (ENS) had highly rich genes.
are defined by the selective expression of a single gene, or by a combination of two or more genes.
same genes can also be shared in different intestinal neuron categories (ENCs) and expressed differently in different intestinal neuron categories (ENCs).
, the researchers linked the new ENS classification to previously discovered characteristics of functional intestinal neuron types.
ENC is generally composed of cells of the same functional type.
, enC1-4 is identified as an excitable motor neuron, while ENC8 and 9 inhibit motor neurons.
genetic map shows several additional neuroactive substances with class-specific expressions.
-output communication of synapses is the basic difference between neuron types and is determined by connections, reactions, and the release of neurotransmitter/peptides.
researchers explored the gene families that control these functions and found differential expressions between ENCs.
reactions to intestinal neurotransmitter/peptides are category specific.
For example, glutamate-induced Grm5 is selectively expressed in ENC7, while growth inhibitors and Npy-subjects Sstr5 and Npy5r are limited to ENC12.ENC6 selectively expressing recent signal transductors/regulators associated with immune cell intestinal control.
ENCs also show unique combinations of adhesive molecules, which may determine their precise connection patterns.
, the selective expression of genes that give neurons the characteristics strongly supports ENC1-12 to represent different neurons in function.
have determined that most families of rich genes can be classified according to their transcription, adhesion, and signal transductivity activity.
proteins involved in the distribution of tissues/channels/vesicle membranes (communication characteristics) contribute to the specific characteristic division of neuron subtypes.
, to verify the presence of different categories of intestinal neurons in the body, the researchers conducted immunoglostification tests on the mice's small intestines.
a total of 23 proteins were analyzed using the newly defined ENCs.
results showed that expression combinations of different proteins could define ENC categories (discrete motor neurons, sensory neurons, and intermediate neurons), such as 5-HT (5-serotonin) being identified in a sub-set of ENC12 neurons.
then, the researchers identified neuron ENC6 and ENC12 subpopons using Nmu-Cre and Cck-IRES-Cre mice with specific markers to exhibit morphological and projected IPAN (endogenous primary incoming neuron) characteristics.
To understand the general and specific differentiation processes of ENC1-12 formation, the researchers analyzed the entire ENS sequence isolated from the small intestine of the Wnt1Cre-R26 tumor at gynaebage E15.5 and E18.5, when most intermuscular neuron types had been generated.
analysis of differences in the expression of these broad cell states found that transcription and signal transductive genes may play a role in stem cell maintenance, neurogenesis, neuron differentiation, SCP, and enteroglia cells.
Notch signal path seems to be closely related to the development of regulating basic ENS.
cells show rich expressions of various chromatin modifiers (Baz1a, Tox3, Bcl11b), which may perform large transcriptional changes associated with the differentiation of progenitic cells into neurons.
analysis of ENS in embryos revealed the principle of diversification after filamentation, where only ENC1 or ENC8 esophageal features were developed through the stem cell nervous system, and other subsype features were formed through subsequent differentiation.
researchers noted that there was a boundary between ENC8/9 and ENC12, indicating that neurons with early ENC8/9 symptoms could be converted into ENC12 neurons, and that Pbx3 expression was associated with transition points.
, in order to assess the role of Pbx3 in ENS development, the study analyzed the intestines of Pbx3 -/- mutant mice.
Pbx3 deficiency did not affect intestinal proliferation or general procedures for nerve development.
PBX3 inhibits ENC8/9 esogeals, allowing or inducing differentiation of ENC12 esopes.
, this study provides conceptual and molecular resources for analyzing the human intestinal nervous system (ENS) circuit, and predicts the key regulatory factors of directional differentiation in different intestinal neuron categories.
also provide theoretical support for the treatment of intestinal diseases.
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