Extended Data Fig. 4: Microbiota-dependent expression of AHR in colonic neurons.
From: Neuronal programming by microbiota regulates intestinal physiology
a, TPM values (mean ± s.d.) for AHR transcripts in neuronal and non-neuronal nuclear preparations from muscularis externa from colon and small intestine of SPF and germ-free mice. n = 4 (SPF) and 3 (germ-free) mice. b, c, Myenteric ganglia immunostained for KIT (which identifies interstitial cells of Cajal) and AHR (b) or SOX10 (enteric glial cells) and AHR (c). AHR+ cells are distinct from intestitial cells of Cajal and enteric glia. Scale bars, 30 μm. d–f, Immunostaining of neurons from the colon of wild-type mice for AHR (d–f) and the neuronal markers peripherin and NF-M (d), calbindin and nNOS (e), and calretinin and HuC/D (f). AHR signal was detected in all subtypes of myenteric neurons (arrowheads). Scale bars, 30 μm. g, Immunostaining of neurons from the colon of ChAT-mCherry-TVA reporter mice for mCherry (red), AHR (green) and HuC/D (blue). Arrowhead indicates an enteric neuron positive for ChAT and AHR. Scale bar, 30 μm. h, i, Immunostaining of myenteric ganglia from the jejunum (h) and ileum (i) with the pan-neuronal marker peripherin (blue) and AHR (red). Scale bar, 30 μm. j, k, Representative images of enteric ganglia from duodenum (j) and colon (k) hybridized with RNAscope probe for Ahr (green). Dotted line defines the borders of myenteric ganglia. Scale bar, 30 μm. l, Quantification (mean ± s.d.) of RNAscope signal per neuron is shown (two-sided non-parametric Mann–Whitney U-test). n = 91 small-intestine and 254 colon neurons from 6 mice. m–o, Immunostaining of ganglia from the colon of control (m), antibiotic-treated (n) and microbiota-colonized, antibiotic-treated (o) mice with peripherin (blue) and AHR (red). Small panels show signal for AHR (top) and peripherin (bottom). n = 3 mice for each condition. Scale bars, 30 μm. p, q, Representative images of enteric ganglia from the colon of control (p) and antibiotic-treated (q) mice hybridized with RNAscope probe for Ahr (green). Dotted line defines the borders of myenteric ganglia and arrows indicate positive cells. Scale bars, 30 μm. r, Quantification (mean ± s.d.) of RNAscope signal per neuron is also shown (two-sided non-parametric Mann–Whitney U-test). n = 518 neurons from 4 control and 468 neurons from 4 antibiotic-treated mice. Data represent two (j, k, m–q) or three (b–i) independent experiments.
