Fig 2: Long term Sonidegib treatment in mouse reduces taste buds (TB) and SHH ligand in circumvallate papilla (CV) whereas proliferation and innervation are retained. Immunofluorescent antibody detection of SHH ligand (red) and K8 (green) for TB cells; K8 (red) for TB cells and Ki67 (green) for cell proliferation; and K8 (red) with NF (green) for GL innervation or P2X3 (green) for GL taste fibers, after Vehicle or 48d Sonidegib treatment. For SHH/K8, large dotted lines indicate the basal lamina. Small dotted lines outline the surface epithelium. Inset (K8/P2X3) shows an image of nerves extending into CV epithelial basal lamina (arrow). Scale bar: 50 µm, applies to all images. Inset at 2×.

Fig 3: Fungiform papillae (FP) and taste buds (TB) on the anterior tongue are innervated by Phox2b-, P2X3- and Shh-positive chorda tympani (CT) nerve fibers. a, a’ H&E staining demonstrates FP and the single apical TB and surrounding FILIF in the stratified squamous anterior tongue epithelium. a’’ Schematic illustration of the FP displays the apical TB and innervation by CT fibers in yellow, with lingual nerve (LN) innervation (not colored) to apical and lateral FP epithelium but not to TB. Yellow cells at the TB base are locations for SHH-positive cells. Blue epithelial basal and stromal cells in the FP denote Gli1 + cells, signaling in the Hedgehog pathway. b, b’ Antibody detection of NF-H (green) in the CT/LN nerve bundles coursing through the anterior tongue and within the core of the FP b’. K8 + cells (red) are TB cells b’. c, c’, c’’ Phox2b + fibers expression (seen with RFP immunoreactions in red) is shown in the anterior tongue and into the core of the FP and K8 + (green) TB cells c’. Antibody detection of P2X3 (green) confirms the CT location in the FP core with Phox2b and within the K8 + (red) TB cells c’’. d, d’, d’’ Antibody detection of P2X3 colabels with the location of Phox2b expression in the CT and TB. e,e,e’’ P2X3 + fibers and cells colabel with Shh (RFP, red, expression in ShhCreER; R26RFP mice) in the FP core and within TB. f, f’, f’’ Shh expression is seen in all FP f and within CT fibers and TB cells f’, f’’. Scale bars are within each image

Fig 4: No change in the number of 5-HT3A-GFP geniculate ganglion neurons in Skn-1a-/- mice. Skn-1a-/- mice were crossed to 5-HT3A-GFP mice. A, B, Geniculate ganglia of Skn-1a-/-/5-HT3A-GFP and WT littermates (still expressing 5-HT3A-GFP) were labeled with antibodies against GFP (green), P2X3 (magenta), and SNAP25 (blue). The number of labeled cells was counted for each label. C, Percent of total cells showing GFP, P2X3, or SNAP25 immunoreactivity. KO of Skn-1a had no effect. D, Percent of total cells showing each combination of labels. KO of Skn-1a had no effect. Data are a summary of from three WT and four KO mice. Bar chart depicts sample mean and error bars show SEM.

Fig 5: Schematic model of purinergic signalling from L-cells to local targets. ATP released together with GLP-1 and PYY from L-cells, exemplified here by angiotensin receptor stimulation, can activate purinergic receptors located on neighbouring enterocytes, potentially modulating fluid secretion by opening of Ca2+-sensitive K+ channels. ATP released from L-cells also targets local nerve terminals of vagal afferent neurons deriving from the nodose ganglion (brown cell) which express purinergic P2X3 receptors, providing ATP neurotransmitter mediated signalling pathway from the intestinal epithelium to the brainstem