senGC maturation is microbiota dependent. (A) Ex vivo mucus growth in adult GF and ConvR mouse colon after stimulation with bacterial MAMPs. (B) Ex vivo mucus growth dose response to P3CSK4 in adult GF and ConvR mouse colon. (C) Ex vivo mucus growth in adult GF and ConvR mouse colon stimulated with P3CSK4 in the presence or absence of senGC activation inhibitors. (D) AB/PAS-stained tissue sections from ex vivo experiments illustrated in A. Emptied upper crypt GCs (red arrowheads) and lower crypt cavitation (yellow arrowheads) indicated. (E) Whole-mount confocal imaging of adult GF mouse colon treated with fluorescent dextran tracer. Images show x/y-axis (upper panel) and x/z-axis (lower panel) cross-sections illustrating dextran uptake by an upper crypt GC (purple arrowhead). (F) Ex vivo mucus growth in neonatal (P3, 5, and 15) and postweaning (P33) rat colon stimulated with P3CSK4 in the presence or absence of Dynasore inhibitor. (G) Standardized expression of genes (columns) encoding known and predicted secreted proteins upregulated in mucus from P9-adult compared with P1–P7 rats (see Fig. 1 H) in GC subpopulations (rows) identified by scRNA-seq. “Secretion” row indicates evidence of secretion determined by prior annotation or in silico predication of classical or nonclassical secretion by SecretomeP. (H) Quantification of the frequency of Tgm3-expressing GCs as a proportion of the total GC population in neonatal (P3, P9, P14, and P19) and postweaning (P24) colonic tissue sections from ConvR mice. (I) Confocal micrographs of representative tissue sections from P3 and P14 ConvR mice stained for Tgm3 (green) or the epithelial border and GC-binding lectin WGA (grey) and the GC-specific lectin UEA1 (red). The epithelial surface (blue dashed line) and an individual GC from each image is indicated (yellow dashed line). Data represent n = 3–5 (A–F, H, and I) animals per group, as indicated. All data are pooled from at least two independent litters or experiments. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by two-way ANOVA and Fisher’s LSD (A and C) or Kruskall–Wallis and uncorrected Dunn’s test (F and H); P < 0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). Image scale bars are 50 µm (D and I) or 20 µm (E). # note: ConvR data displayed in A and B are reproduced from our previous publication (Birchenough et al., 2016) and are shown for illustrative purposes only.
Figure 4.

senGC maturation is microbiota dependent. (A) Ex vivo mucus growth in adult GF and ConvR mouse colon after stimulation with bacterial MAMPs. (B) Ex vivo mucus growth dose response to P3CSK4 in adult GF and ConvR mouse colon. (C) Ex vivo mucus growth in adult GF and ConvR mouse colon stimulated with P3CSK4 in the presence or absence of senGC activation inhibitors. (D) AB/PAS-stained tissue sections from ex vivo experiments illustrated in A. Emptied upper crypt GCs (red arrowheads) and lower crypt cavitation (yellow arrowheads) indicated. (E) Whole-mount confocal imaging of adult GF mouse colon treated with fluorescent dextran tracer. Images show x/y-axis (upper panel) and x/z-axis (lower panel) cross-sections illustrating dextran uptake by an upper crypt GC (purple arrowhead). (F) Ex vivo mucus growth in neonatal (P3, 5, and 15) and postweaning (P33) rat colon stimulated with P3CSK4 in the presence or absence of Dynasore inhibitor. (G) Standardized expression of genes (columns) encoding known and predicted secreted proteins upregulated in mucus from P9-adult compared with P1–P7 rats (see Fig. 1 H) in GC subpopulations (rows) identified by scRNA-seq. “Secretion” row indicates evidence of secretion determined by prior annotation or in silico predication of classical or nonclassical secretion by SecretomeP. (H) Quantification of the frequency of Tgm3-expressing GCs as a proportion of the total GC population in neonatal (P3, P9, P14, and P19) and postweaning (P24) colonic tissue sections from ConvR mice. (I) Confocal micrographs of representative tissue sections from P3 and P14 ConvR mice stained for Tgm3 (green) or the epithelial border and GC-binding lectin WGA (grey) and the GC-specific lectin UEA1 (red). The epithelial surface (blue dashed line) and an individual GC from each image is indicated (yellow dashed line). Data represent n = 3–5 (A–F, H, and I) animals per group, as indicated. All data are pooled from at least two independent litters or experiments. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by two-way ANOVA and Fisher’s LSD (A and C) or Kruskall–Wallis and uncorrected Dunn’s test (F and H); P < 0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). Image scale bars are 50 µm (D and I) or 20 µm (E). # note: ConvR data displayed in A and B are reproduced from our previous publication (Birchenough et al., 2016) and are shown for illustrative purposes only.

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