Specialized Mechanosensory Epithelial Cells in Mouse Gut Intrinsic Tactile Sensitivity

Abstract

Background and aims: The gastrointestinal (GI) tract extracts nutrients from ingested meals while protecting the organism from infectious agents frequently present in meals. Consequently, most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of the its external interface to optimize its function. Specialized sensory enteroendocrine cells (EECs) in GI epithelium interact intimately with luminal contents. A subpopulation of EECs express the mechanically gated ion channel Piezo2 and are developmentally and functionally like the skin's touch sensor- the Merkel cell. We hypothesized that Piezo2+ EECs endow the gut with intrinsic tactile sensitivity.

Methods: We generated transgenic mouse models with optogenetic activators in EECs and Piezo2 conditional knockouts. We used a range of reference standard and novel techniques from single cells to living animals, including single-cell RNA sequencing and opto-electrophysiology, opto-organ baths with luminal shear forces, and in vivo studies that assayed GI transit while manipulating the physical properties of luminal contents.

Results: Piezo2+ EECs have transcriptomic features of synaptically connected, mechanosensory epithelial cells. EEC activation by optogenetics and forces led to Piezo2-dependent alterations in colonic propagating contractions driven by intrinsic circuitry, with Piezo2+ EECs detecting the small luminal forces and physical properties of the luminal contents to regulate transit times in the small and large bowel.

Conclusions: The GI tract has intrinsic tactile sensitivity that depends on Piezo2+ EECs and allows it to detect luminal forces and physical properties of luminal contents to modulate physiology.

Keywords: Enteroendocrine Cell; GI Physiology; Ion Channels; Mechanosensitivity; Neuroepithelial Connection.

Figure 1.

Transcriptional profile of the epithelial Piezo2 enteroendocrine cell subpopulation. (A) Strategy to enrich Piezo2+ cells for single-cell RNA sequencing (scRNAseq). Lineage-traced cells from Piezo2^tomato colon mucosa: tomato+ if ever expressed Piezo2 (red) and some continued to express Piezo2 (red/blue). Tomato+ cells were sorted by fluorescence-activated cell sorting (FACS) and examined by scRNAseq. (B) Representative epifluorescence micrograph of Piezo2^tomato colon showing tomato+ structures and cells: ^&myenteric plexus, ^#intracrypt, and *sporadic epithelial cells. Red line shows mucosal peeling plane. (C) FACS sorting of dissociated cells from Piezo2^tomato colonic mucosa showing tomato+ cell population (from total mean ± SEM, 1,522,111 ± 142,606 cells, tomato+ were 0.2%−0.9%, mean ± SEM, 0.5% ± 0.2%, n = 3). (D) t-distributed stochastic neighbor embedding (tSNE) plot of 10 tomato+ populations. (E) tSNE plot of tomato+ subpopulations showing 3 clusters that actively express Piezo2. (F) Heatmap showing top cluster defining genes and Piezo2 expression across clusters (bottom strip). (G) Epithelial Piezo2+ population expresses EEC-specific developmental factors, a range of chemo- and olfactory receptors signal transduction ion channels, signaling and synaptic molecules, active zone, neuroepithelial communication, and post-synaptic receptors. (H) Pseudotime plot of the Piezo2+ enterochromaffin cell population showing that from root state (cyan), Piezo2 cells segregate into the following 3 states: Piezo2^loTph1^hi, Piezo2^hiTph1^hi, and Piezo2^hiTph1^lo. (I) Comparing transcriptomes of the Piezo2^hi to Piezo2^lo populations suggests that Piezo2^hi is an electrically excitable synaptically connected EEC subpopulation. (J) Colonic organoids from Piezo2^tomato mice show tomato+ cells (magenta). Scale bars: 50 μm (top), 10 μm (bottom). (K) FACS of dissociated Piezo2^tomato colonic organoids. (I) Quantitative polymerase chain reaction of FAC sorted organoid-derived Piezo2^tomato epithelial cells showing they are EECs enriched in Chga, Tph1, and Piezo2. (M) Piezo2 EECs are a unique population of synaptically connected electrically active mechanosensory EECs. Piezo2 epithelial cells are defined by EEC transcription factors (NeuroD1, Insm1, and Nkx2-2) and enriched in receptors for mechanical forces (Piezo2), and metabolites (Olfr78, Ffar2 [short-chain fatty acids]), and signal transduction ion channels (Scn3a, Cacna1h, Cacna1a). Synaptic vesicles (Chga/b, Snap25, Syt7) contain neurotransmitters, especially serotonin (Tph1), and less so others (secretin [Sct], substance P [Tac1], Glp-1 [Gcg and Pcsk1], Pyy, and Somatostatin [Sst]). They are enriched in molecules that form a presynaptic active zone (Pclo, Rimbp2, and Rim2), but also post-synaptic receptors (glutamate [Grin3a]).

Figure 2.

EEC^ReaChR cells in the GI tract are EECs that are predominately enterochromaffin cells that express a red light–gated ion channel ReaChR. (A) Model where Piezo2 and ReaChR are expressed by EECs, and activation of EECs by force or red-light releases 5-HT to enteric neurons that control GI physiologic functions. (B–J) Projection of confocal stack images of EEC^ReaChR mouse colon with transgenic ReaChR expression (magenta in panels B, E, and H) colocalizing with immunofluorescence of EEC marker CgA (green in panels C and D), 5-HT (green in panels F and G), and Piezo2 (green in panels I and J). Nuclei counterstained with 4′,6-diamidino-2-phenylindole (blue). Lower panels expanded from within white rectangles. Scale bars = 20 μm. (B) ReaChR expression (magenta), (C) CgA immunofluorescence (green) and (D) colocalization. (E) ReaChR expression (magenta), (F) 5-HT immunofluorescence (green) and (G) colocalization. (H) ReaChR expression (magenta), (I) Piezo2 immunofluorescence (green) and (J) colocalization.

Figure 3.

Optical and mechanical stimulation activate single EEC^ReaChR cells. (A) Projection of a confocal stack overlayed with DIC of live 3-dimensional colonic organoid derived from EEC^ReaChR mouse with ReaChR (red) channel and transmitted light (gray). (B) Experimental setup showing an organoid-derived primary EEC under whole-cell voltage clamp electrophysiology and stimulated with red light. (C) Representative optically induced whole-cell currents with voltage-clamped electrophysiology. Scale bars = ordinate, 50 pA, abscissa, 1 second. (D) Normalized current dose-duration curve of EEC^ReaChR cell. (E) Current–voltage relationship (I/V) of peak current in EEC^ReaChR cell. Scale bars = ordinate, 200 pA, abscissa, 600 milliseconds. (F) Experimental setup showing mechanical or optogenetic stimulation of EEC^ReaChR cell (red) leading to 5-HT release that is detected and reported by the 5-HT biosensor. (G) Confocal stack image showing EEC^ReaChR cell (red) co-cultured with 5-HT biosensor (5HT3R/R-GECO, green). Scale bar = 20 μm. (H) Representative 5-HT biosensor fluorescence (ΔF/F₀) traces after EEC^ReaChR activation by light (black), block by 5HT₃R inhibitor ondansetron (OND, 0.1 μM, red), and mechanical stimulation (blue). Stimulation period in the line below trace. Scale bars = ordinate, 0.5ΔF/F₀, abscissa, 30 seconds. (I) Average 5-HT biosensor responses to EEC^ReaChR light stimulation (black), with OND (red), and with mechanical stimulation (blue) (individual data points paired, and bars showing mean ± SEM, n = 9, paired t test). (J) Representative 5-HT biosensor fluorescence (ΔF/F₀) traces after EEC activation with light in the presence of Piezo2 blocker Gd³⁺ (blue) mechanical stimulation with Gd³⁺ (red) and mechanical stimulation in washed control bath solution (blue). Stimulation period in the line below trace. Scale bars = ordinate, 0.5ΔF/F₀, abscissa, 30 seconds. (K) Average 5-HT biosensor response to EEC^ReaChR red-light stimulation with Gd³⁺ (purple) mechanical stimulation with Gd³⁺ (green) and washout control mechanical stimulation (blue) (mean ± SEM, n = 15, paired t test). **P < .01, ****P < .0001.

Figure 4.

Optical stimulation of EEC^ReaChR increases colonic propagating contraction frequency. (A) Experimental setup with organ bath used to measure optically stimulated (λ) propagating contractions through oral (ΔT₁) and aboral (ΔT₂) tension transducers and aboral pressure (ΔP) sensor. (B) Representative traces (ΔT₁, ΔT₂, ΔP) showing baseline, optical stimulation (2Hz, 40% duty cycle, 10 seconds, 90 seconds start to start), and recovery contraction frequency in EEC^ReaChR colon. Gray dashed line at peak of each tension trace (from ΔT1 to ΔT2) follows contraction propagating along the tissue. Optical stimulation shown between T₁ and T₂ traces (red). (C) Average propagating contraction frequency during baseline (gray), optical stimulation (red), and recovery (blue) in EEC^WT and EEC^ReaChR experimental colons (mean ± SEM, EEC^WT n = 6, EEC^ReaChR n = 14, paired t test) (ΔT1 scale bars = ordinate, 0.5 mN, abscissa, 1 minute; ΔT2 scale bars = ordinate, 1 mN, abscissa, 1 minute; and ΔP scale bars = ordinate, 5 cmH₂O, abscissa, 1 minute). **P < .01, ***P < .001.

Figure 5.

Mechanosensitive EEC activation by small luminal forces increases colonic contraction frequency. (A) Experimental setup to measure luminal shear (black arrow in the lumen) induced changes in colonic motility reported by a tension transducer (ΔT₁). (B) Contractions intervals, displayed as black ticks, in Piezo2^WT colon basal and intraluminal shear (small force: 10 mL/h and large force: 25 mL/h, for 10 seconds, 90 seconds start to start). (C) Contraction frequency during baseline, intraluminal, and recovery in Piezo2^WT colon (mean ± SEM, 10 mL/h, n = 12; 25 mL/h, n = 6, paired t test). (D) Contractions intervals, displayed as red ticks, in Piezo2^CKO colon at rest and with intraluminal shear flow (small force: 10 mL/h and large force: 25 mL/h, 10 seconds, 90 seconds start to start). (E) Contraction frequency during baseline, intraluminal flow, and recovery in Piezo2^CKO colon (mean ± SEM, 10 mL/h, n = 10; 25 mL/h n = 7, paired t test). (F) Percent increase contraction frequency of Piezo2^WT and Piezo2^CKO colon from baseline to shear (mean ± SEM, Piezo2^WT n = 6, Piezo2^CKO n = 7, 1-tailed t test). (G) Experimental organ bath set up to measure luminal flow (black arrow in lumen) and 625 nm red-light optogenetic (2 Hz, 40% duty cycle) induced changes in colonic motility through a tension transducer (ΔT1). (H) Contraction intervals of tension traces showing basal, luminal flow (10 mL/h) baseline, flow (10 mL/h), and optogenetic stimulation with flow (2 Hz, 40% duty cycle and 10 mL/h) frequency in the presence of luminal Gd³⁺ in EEC^WT (top) and EEC^ReaChR (bottom) colon. (I) Contraction frequency of EEC^WT and EEC^ReaChR colon during baseline, intraluminal flow, baseline with Gd³⁺, Gd³⁺ intraluminal flow, and Gd³⁺ flow with optogenetic stimulation (mean ± SEM, EEC^WT n = 6, EEC^ReaChR n = 7, paired t test). *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 6.

Mechanosensitive Piezo2 EECs are regulators of tactile sensing by the luminal gut. (A) In vivo assays to determine the role of Piezo2 EECs in the gut: whole-gut transit (red), gastric emptying (blue), small intestine (yellow), and colon (purple). (B) Whole-gut transit time for Piezo2^WT and Piezo2^CKO (mean ± SEM, n = 12, n = 28, unpaired t test). (C) Gastric emptying T_1/2 was not different between Piezo2^CKO and Piezo2^WT mice (mean ± SEM, n = 5, n = 7, unpaired t test). (D) Top row, small bowel transit in Piezo2^WT vs Piezo2^CKO for liquids. Bottom row, small bowel transit for 390 μm fluorescent beads (n = 8, n = 6, unpaired t test). Spectral analysis of fluorescence deconstructed by frequency, showing the dominant frequencies of the signa (scale bar = 0.1). (E) Piezo2^WT (left) and Piezo2^CKO (right) pellet length and width, with image of pellets comparing size (mean ± SEM, n = 40, n = 71, unpaired t test) (scale bar = 2.0 mm). (F) Distal colonic transit of Piezo2^WT and Piezo2^CKO mice in 1 mm, 2 mm, and 3 mm beads (mean ± SEM, unpaired t test). *P < .05, **P < .01.