Enteroendocrine cell lineages that differentially control feeding and gut motility
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As digested food moves through the intestines specialized epithelial cells (called Enterochromaffin Cells or EECs) sense and respond to the constituent chemicals. The current work utilizes single-cell transcriptomic analyses and intersectional approaches to define and genetically manipulate subsets of EECs. Key findings are that direct stimulation of EEC subtypes influences key aspects of feeding, specifically gut transit, ingestion, and food preference.
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Abstract
Enteroendocrine cells are specialized sensory cells of the gut-brain axis that are sparsely distributed along the intestinal epithelium. The functions of enteroendocrine cells have classically been inferred by the gut hormones they release. However, individual enteroendocrine cells typically produce multiple, sometimes apparently opposing, gut hormones in combination, and some gut hormones are also produced elsewhere in the body. Here, we developed approaches involving intersectional genetics to enable selective access to enteroendocrine cells in vivo in mice. We targeted FlpO expression to the endogenous Villin1 locus (in Vil1-p2a-FlpO knock-in mice) to restrict reporter expression to intestinal epithelium. Combined use of Cre and Flp alleles effectively targeted major transcriptome-defined enteroendocrine cell lineages that produce serotonin, glucagon-like peptide 1, cholecystokinin, somatostatin, or glucose-dependent insulinotropic polypeptide. Chemogenetic activation of different enteroendocrine cell types variably impacted feeding behavior and gut motility. Defining the physiological roles of different enteroendocrine cell types provides an essential framework for understanding sensory biology of the intestine.
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eLife assessment
As digested food moves through the intestines specialized epithelial cells (called Enterochromaffin Cells or EECs) sense and respond to the constituent chemicals. The current work utilizes single-cell transcriptomic analyses and intersectional approaches to define and genetically manipulate subsets of EECs. Key findings are that direct stimulation of EEC subtypes influences key aspects of feeding, specifically gut transit, ingestion, and food preference.
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Reviewer #1 (Public Review):
Of course, many of the most important aspects of feeding happen post-ingestion. As digested food moves through the intestines specialized epithelial cells (called Enterochromaffin Cells or EECs) sense and respond to the constituent chemicals. The function of EECs initiates physiological responses to facilitate nutrient absorption, protect from toxins and encourage proper waste removal. EECs are sparse and heterogenous and release a variety of transmitters and diffusible signaling molecules that signal to peripheral neurons and the brain. Their collective activity slows or speeds gut transit and promotes feelings of satiety or malaise. The current work by Liberles and colleagues seeks to provide deeper insight into the function of EECs. They build on previous work by further categorizing these cells by their …
Reviewer #1 (Public Review):
Of course, many of the most important aspects of feeding happen post-ingestion. As digested food moves through the intestines specialized epithelial cells (called Enterochromaffin Cells or EECs) sense and respond to the constituent chemicals. The function of EECs initiates physiological responses to facilitate nutrient absorption, protect from toxins and encourage proper waste removal. EECs are sparse and heterogenous and release a variety of transmitters and diffusible signaling molecules that signal to peripheral neurons and the brain. Their collective activity slows or speeds gut transit and promotes feelings of satiety or malaise. The current work by Liberles and colleagues seeks to provide deeper insight into the function of EECs. They build on previous work by further categorizing these cells by their unique gene expression signatures. The work utilizes single-cell transcriptomic analyses and intersectional approaches to define and genetically manipulate subsets of EECs. A key aspect of the study is behavioral assays used to investigate how direct stimulation of EEC subtypes influences key aspects of feeding, specifically gut transit, ingestion, and food preference.
The work has several strengths. A new mouse line (Villin-flp) is developed and used intersectionally with Cre mouse lines to manipulate different subsets of epithelial cells. The authors characterize these compound mouse strains and how the labeled cells map onto transcriptomic class. These data are reasonably comprehensive and show the exclusion of marker expression from the central nervous system, important controls. The chemogenetic activation strategy is an elegant way to probe the consequences of EEC stimulation by Gq coupled GPCR signalling. The gut transit experiments show clear effects.
The weakness is it remains unclear whether stimulation of the DREADD receptor outside the intestinal EECs really has consequences (e.g. in the tongue), the behaviors tested are somewhat limited, the responses to CNO administration variable between animals, and the effect sizes are small.
Overall, this is an interesting study and provides useful tools for the field.
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Reviewer #2 (Public Review):
Enteroendocrine cells (EEC) line the gut and prior evidence suggest that they are primary sensors of gut contents. In turn, these cells release transmitters that regulate gut function, including gut motility, enzyme secretion, and gut permeability. More recent studies have also found synaptic connections between EEC and neural sensory fibers that connect the gut to the brain, implicating this pathway in taste learning. Thus, EEC signals can be integrated with sensory signals originating in more distal areas of the alimentary canal.
EECs express a variety of receptors and transmitters that are hypothesized to contribute to the diversity of sensing and motor functions. In this report, Hayashi et al develop a novel transgenic mouse that permits manipulation of EEC subtypes via intersectional methods. Using this …
Reviewer #2 (Public Review):
Enteroendocrine cells (EEC) line the gut and prior evidence suggest that they are primary sensors of gut contents. In turn, these cells release transmitters that regulate gut function, including gut motility, enzyme secretion, and gut permeability. More recent studies have also found synaptic connections between EEC and neural sensory fibers that connect the gut to the brain, implicating this pathway in taste learning. Thus, EEC signals can be integrated with sensory signals originating in more distal areas of the alimentary canal.
EECs express a variety of receptors and transmitters that are hypothesized to contribute to the diversity of sensing and motor functions. In this report, Hayashi et al develop a novel transgenic mouse that permits manipulation of EEC subtypes via intersectional methods. Using this approach, they identify differential roles for EEC subtypes in controlling gut motility and taste learning.
Strengths
• The authors supplement existing single-cell RNA sequencing of the proximal intestine.
• A Vil1-2a-Flp mouse was generated, which exhibits highly selective expression in the gut epithelium. This mouse line can be used to manipulate EEC subtypes when bred with other Cre driver lines and double conditional (Flp/Cre) mice.
• Using the above tool, different EEC subtypes were histologically characterized along the alimentary canal. Additionally, other tissues were examined, including the brain, pancreas, and lungs to demonstrate the gut specificity of their approach. The intersectional approach yield sparse recombination in the pancreas, therefore the authors included controls in their gut motility and feeding studies to account for this.
• In probing the function of distinct EECs, it was found that Cck(cholecystokinin) and Gcg (GLP-1) expressing EECs slow down gut motility, whereas Tac1 (substance P) and Pet1(serotonin) expressing cells increase motility.
• Food intake studies revealed several subpopulations that decrease feeding (Pet1, Npy1r, Cck, Gcg).
• A conditioned flavor preference assay suggests that some of the above EEC subtypes (Pet1, Tac1, Npy1r, Gcg) decrease feeding in part through conditioned flavor avoidance. -
Reviewer #3 (Public Review):
This manuscript describes a villin-2a-Flp-based intersectional strategy for selectively targeting EEC in the intestine and uses it to examine the function of subsets. The approach for targeting select subsets of enteroendocrine cells described here will be important for neuroscientists, endocrinologists, microbiologists, and other scientists studying nutritional biology. Here single-cell sequencing is used, primarily, to confirm what was already known about EEC classes at a transcriptomic level. The intersectional approach described here has the potential to provide broad access to EECs. However, from the relatively limited characterization of targeted EEC cells, it appears that the genes that have been combined with the villin driver largely fail to selectively target transcriptomically defined cell types. …
Reviewer #3 (Public Review):
This manuscript describes a villin-2a-Flp-based intersectional strategy for selectively targeting EEC in the intestine and uses it to examine the function of subsets. The approach for targeting select subsets of enteroendocrine cells described here will be important for neuroscientists, endocrinologists, microbiologists, and other scientists studying nutritional biology. Here single-cell sequencing is used, primarily, to confirm what was already known about EEC classes at a transcriptomic level. The intersectional approach described here has the potential to provide broad access to EECs. However, from the relatively limited characterization of targeted EEC cells, it appears that the genes that have been combined with the villin driver largely fail to selectively target transcriptomically defined cell types. Thus, at present, this manuscript fails to convincingly target transcriptome-defined enteroendocrine cell types, and conclusions on gut motility, feeding behavior, and flavor avoidance are overstated.
Some aspects of the study are compelling including the use of villin drivers as a means to restrict recombination to the epithelium containing EECs. The single-cell data (although not unique to this study) proved a basis for a better understanding of EECs and also their developmental specification. The charcoal-based gut motility assay appears valuable (although the results are perhaps not surprising given what was already known). In addition, some of the care taken characterizing extra-EEC expression is commendable. However, the manuscript is difficult to read with important details scattered in different figures and text (e.g., the characterization of expression patterns of the various lines). Moreover, whereas some things like the genetic makeup of the lines are always specified in full (excruciating) details, the expression patterns of the various lines are often casually dealt with e.g., describing separate targeting of L and I cells despite no evidence that this is actually being done. I would hope that the authors will address these issues and devote significant attention to making the paper more accessible to its readers.
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