A branching model of lineage differentiation underpinning the neurogenic potential of enteric glia.

Glial cells have been proposed as a source of neural progenitors, but the mechanisms underpinning the neurogenic potential of adult glia are not known. Using single cell transcriptomic profiling, we show that enteric glial cells represent a cell state attained by autonomic neural crest cells as they transition along a linear differentiation trajectory that allows them to retain neurogenic potential while acquiring mature glial functions. Key neurogenic loci in early enteric nervous system progenitors remain in open chromatin configuration in mature enteric glia, thus facilitating neuronal differentiation under appropriate conditions. Molecular profiling and gene targeting of enteric glial cells in a cell culture model of enteric neurogenesis and a gut injury model demonstrate that neuronal differentiation of glia is driven by transcriptional programs employed in vivo by early progenitors. Our work provides mechanistic insight into the regulatory landscape underpinning the development of intestinal neural circuits and generates a platform for advancing glial cells as therapeutic agents for the treatment of neural deficits.

Molecular profiling of enteric nervous system cell lineages.

The enteric nervous system (ENS) is an extensive network of enteric neurons and glial cells that is intrinsic to the gut wall and regulates almost all aspects of intestinal physiology. While considerable advancement has been made in understanding the genetic programs regulating ENS development, there is limited understanding of the molecular pathways that control ENS function in adult stages. One of the limitations in advancing the molecular characterization of the adult ENS relates to technical difficulties in purifying healthy neurons and glia from adult intestinal tissues. To overcome this, we developed novel methods for performing transcriptomic analysis of enteric neurons and glia, which are based on the isolation of fluorescently labeled nuclei. Here we provide a step-by-step protocol for the labeling of adult mouse enteric neuronal nuclei using adeno-associated-virus-mediated gene transfer, isolation of the labeled nuclei by fluorimetric analysis, RNA purification and nuclear RNA sequencing. This protocol has also been adapted for the isolation of enteric neuron and glia nuclei from myenteric plexus preparations from adult zebrafish intestine. Finally, we describe a method for visualization and quantification of RNA in myenteric ganglia: Spatial Integration of Granular Nuclear Signals (SIGNS). By following this protocol, it takes ~3 d to generate RNA and create cDNA libraries for nuclear RNA sequencing and 4 d to carry out high-resolution RNA expression analysis on ENS tissues.

Regulation of intestinal immunity and tissue repair by enteric glia.

Tissue maintenance and repair depend on the integrated activity of multiple cell types1. Whereas the contributions of epithelial2,3, immune4,5 and stromal cells6,7 in intestinal tissue integrity are well understood, the role of intrinsic neuroglia networks remains largely unknown. Here we uncover important roles of enteric glial cells (EGCs) in intestinal homeostasis, immunity and tissue repair. We demonstrate that infection of mice with Heligmosomoides polygyrus leads to enteric gliosis and the upregulation of an interferon gamma (IFNγ) gene signature. IFNγ-dependent gene modules were also induced in EGCs from patients with inflammatory bowel disease8. Single-cell transcriptomics analysis of the tunica muscularis showed that glia-specific abrogation of IFNγ signalling leads to tissue-wide activation of pro-inflammatory transcriptional programs. Furthermore, disruption of the IFNγ-EGC signalling axis enhanced the inflammatory and granulomatous response of the tunica muscularis to helminths. Mechanistically, we show that the upregulation of Cxcl10 is an early immediate response of EGCs to IFNγ signalling and provide evidence that this chemokine and the downstream amplification of IFNγ signalling in the tunica muscularis are required for a measured inflammatory response to helminths and resolution of the granulomatous pathology. Our study demonstrates that IFNγ signalling in enteric glia is central to intestinal homeostasis and reveals critical roles of the IFNγ-EGC-CXCL10 axis in immune response and tissue repair after infectious challenge.

The enteric nervous system in gastrointestinal disease etiology.

A highly conserved but convoluted network of neurons and glial cells, the enteric nervous system (ENS), is positioned along the wall of the gut to coordinate digestive processes and gastrointestinal homeostasis. Because ENS components are in charge of the autonomous regulation of gut function, it is inevitable that their dysfunction is central to the pathophysiology and symptom generation of gastrointestinal disease. While for neurodevelopmental disorders such as Hirschsprung, ENS pathogenesis appears to be clear-cut, the role for impaired ENS activity in the etiology of other gastrointestinal disorders is less established and is often deemed secondary to other insults like intestinal inflammation. However, mounting experimental evidence in recent years indicates that gastrointestinal homeostasis hinges on multifaceted connections between the ENS, and other cellular networks such as the intestinal epithelium, the immune system, and the intestinal microbiome. Derangement of these interactions could underlie gastrointestinal disease onset and elicit variable degrees of abnormal gut function, pinpointing, perhaps unexpectedly, the ENS as a diligent participant in idiopathic but also in inflammatory and cancerous diseases of the gut. In this review, we discuss the latest evidence on the role of the ENS in the pathogenesis of enteric neuropathies, disorders of gut-brain interaction, inflammatory bowel diseases, and colorectal cancer.

Neuronal programming by microbiota regulates intestinal physiology.

Neural control of the function of visceral organs is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence, and is often dysregulated in gastrointestinal disorders1. Luminal factors, such as diet and microbiota, regulate neurogenic programs of gut motility2-5, but the underlying molecular mechanisms remain unclear. Here we show that the transcription factor aryl hydrocarbon receptor (AHR) functions as a biosensor in intestinal neural circuits, linking their functional output to the microbial environment of the gut lumen. Using nuclear RNA sequencing of mouse enteric neurons that represent distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles that are controlled by the combined effects of host genetic programs and microbial colonization. Microbiota-induced expression of AHR in neurons of the distal gastrointestinal tract enables these neurons to respond to the luminal environment and to induce expression of neuron-specific effector mechanisms. Neuron-specific deletion of Ahr, or constitutive overexpression of its negative feedback regulator CYP1A1, results in reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice. Finally, expression of Ahr in the enteric neurons of mice treated with antibiotics partially restores intestinal motility. Together, our experiments identify AHR signalling in enteric neurons as a regulatory node that integrates the luminal environment with the physiological output of intestinal neural circuits to maintain gut homeostasis and health.

The enteric glia: identity and functions.

Enteric glial cells were first described at the end of the 19th century, but they attracted more interest from researchers only in the last decades of the 20th. Although, they have a different embryological origin, the enteric GLIA share many characteristics with astrocytes, the main glial cell type of the central nervous system (CNS), such as in their expression of the same markers and in their functions. Here we review the construction of the enteric nervous system (ENS), with a focus on enteric glia, and also the main studies that have revealed the action of enteric glia in different aspects of gastrointestinal tract homeostasis, such as in the intestinal barrier, in communications with neurons, and in their action as progenitor cells. We also discuss recent discoveries about the roles of enteric glia in different disorders that affect the ENS, such as degenerative pathologies including Parkinson's and prion diseases, and in cases of intestinal diseases and injury.