Gastrointestinal (GI) motility and defecation are absolute prerequisites for nutrient absorption, fecal elimination and overall health. Normal GI motility, vascular perfusion, and intestinal inflammation are coordinated by vast numbers of neurons that reside within ganglia of the enteric nervous system (ENS) intrinsic to the gut wall. While recent work has identified diverse genes that direct the initial development of progenitor cells that give rise to enteric neurons in the wall of the intestine, we know very little about the genes that are expressed in adult enteric neurons. Consequently we are unable to determine whether efforts to generate enteric neurons produce the normal complement of cell types. Moreover we do not fully understand how distinct types of neurons contribute to overall coordination of intestinal motility because the use of common immunohistochemical markers alone does not distinguish functionally distinct subtypes. As a result, our abilities to target and functionally manipulate specific types of neurons in the gut are extremely limited. To surpass these limitations, our application proposes to develop a comprehensive, single cell transcriptome map of adult enteric neurons in normal mice in parallel with deep sequencing of enteric ganglia from distinct regions of human intestine so that a global gene expression atlas of human enteric ganglia is obtained. To capture mouse enteric neurons for single cell RNA-Seq we will use a fluorescent transgenic mouse line that we developed for live-cell imaging of enteric neurons. Human enteric ganglia will be collected by laser capture microdissection from adult surgical remnants. Comparison of enteric neuron expression profiles between mouse and human data sets will identify conserved genes that mark distinct neuronal subtypes. To set the stage for relating specific neuronal subtypes in the mouse to GI motility we will concurrently quantify intestinal transit and motility patterns across inbred strains of mice using novel ex vivo motility imaging methods. The resulting atlas of molecular fingerprints for enteric neurons in combination with high resolution motility patterns will provide essential information needed to begin targeted, functional manipulation of GI motility in distinct regions of the intestine.
To develop pharmaceuticals and electrode arrays for manipulation of gastrointestinal motility, we need to know all the genes expressed by neurons in the adult enteric nervous system and be able to relate changes in types of neurons to altered intestinal contractility. The proposed work will use the latest revolutionary sequencing technology to catalog genes expressed by enteric neurons of mice and man for comparison to variability in intestinal motility among strains of mice that are genetically distinct. This information is crucial so that scientists can relate different types of neurons to distinct patterns of intestinal contractility and begin to specifically manipulate discrete cell types and thus modulate intestinal function.
| May-Zhang, Aaron A; Deal, Karen K; Southard-Smith, E Michelle (2018) Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue. J Vis Exp : |
