Microbiota induction of senGC maturation via regulation of Duox2. (A) Schematic of the senGC activation pathway highlighting known (black) and putative (red) pathway genes. (B) Expression of known and putative senGC genes in FACS-isolated colonic GCs and colonocytes determined by DESeq2 analysis of bulk RNA sequencing data. (C) Comparison of gene expression ratios between P22 ConvR:GF mice and adult 3-wk ConvD:GF mice quantified by DESeq2 analysis of bulk colonic RNA sequencing data. Genes significantly upregulated (red) or downregulated (blue) by microbiota exposure in both P22 and ConvD mice are indicated. (D) Proportion of unique and shared genes significantly regulated by microbiota exposure in P22 ConvR and adult 3-wk ConvD mice, based on data shown in C. (E) Comparison of microbiota-dependent expression of known and putative senGC activation pathway genes (A and B) in P22 ConvR and adult 3-wk ConvD mice. Subset of data shown in C. Genes not significantly regulated by microbiota in either group (grey) or genes regulated in either P22 ConvR (purple), adult ConvD (yellow), or both groups (teal) are indicated. (F) Relative expression (compared with GF) of Duox2 (left) and Nox1 (right) genes in ConvD (brown) and B. fragilis monoassociated (blue) mice from 1 to 4 wk (w) colonization. Expression determined by qRT-PCR of colonic RNA, normalized to Gapdh and Rplp0 expression. (G) Expression of Duox2 (left) and Nox1 (right) genes in postnatal ConvR (purple; P3–33) and GF (teal; P9–P33) determined by DESeq2 analysis of bulk colonic RNA sequencing data. (H) Confocal micrographs of fixed colonic tissue sections from ConvR WT mice stained for Duox2 (left) and Nox1 (right) mRNA by in situ RNA hybridization and counterstained by Epcam (grey). Duox2- or Nox1-expressing crypt regions are indicated (yellow arrowheads). (I) Confocal micrographs showing upper crypt GCs in fixed colonic tissue sections from ConvR, GF, and ConvD mice stained for Duox2 (red), mucus (UEA1; green), actin (grey), and DNA (blue). Intracellular Duox2 in GCs are indicated (yellow arrowheads). (J) Ex vivo mucus growth in Duox2fl/fl and Duox2ΔIEC colon tissue treated with carbachol (CCh), LPS, or P3CSK4. (K) Ex vivo mucus growth in WT colon tissue treated with P3CSK4 in the presence or absence of the Nox1 inhibitor ML171. Data represent n = 2–5 animals per group, as indicated. All data are pooled from at least two independent experiments or litters. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by DESeq2 (B, C, and E), Kruskal–Wallis and Dunn’s multiple comparison (F), or two-way ANOVA and Fisher’s LSD (J and K); P < 0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). Scale bars are 50 µm (H) or 5 µm (I). FC, fold change.
Figure 7.

Microbiota induction of senGC maturation via regulation of Duox2. (A) Schematic of the senGC activation pathway highlighting known (black) and putative (red) pathway genes. (B) Expression of known and putative senGC genes in FACS-isolated colonic GCs and colonocytes determined by DESeq2 analysis of bulk RNA sequencing data. (C) Comparison of gene expression ratios between P22 ConvR:GF mice and adult 3-wk ConvD:GF mice quantified by DESeq2 analysis of bulk colonic RNA sequencing data. Genes significantly upregulated (red) or downregulated (blue) by microbiota exposure in both P22 and ConvD mice are indicated. (D) Proportion of unique and shared genes significantly regulated by microbiota exposure in P22 ConvR and adult 3-wk ConvD mice, based on data shown in C. (E) Comparison of microbiota-dependent expression of known and putative senGC activation pathway genes (A and B) in P22 ConvR and adult 3-wk ConvD mice. Subset of data shown in C. Genes not significantly regulated by microbiota in either group (grey) or genes regulated in either P22 ConvR (purple), adult ConvD (yellow), or both groups (teal) are indicated. (F) Relative expression (compared with GF) of Duox2 (left) and Nox1 (right) genes in ConvD (brown) and B. fragilis monoassociated (blue) mice from 1 to 4 wk (w) colonization. Expression determined by qRT-PCR of colonic RNA, normalized to Gapdh and Rplp0 expression. (G) Expression of Duox2 (left) and Nox1 (right) genes in postnatal ConvR (purple; P3–33) and GF (teal; P9–P33) determined by DESeq2 analysis of bulk colonic RNA sequencing data. (H) Confocal micrographs of fixed colonic tissue sections from ConvR WT mice stained for Duox2 (left) and Nox1 (right) mRNA by in situ RNA hybridization and counterstained by Epcam (grey). Duox2- or Nox1-expressing crypt regions are indicated (yellow arrowheads). (I) Confocal micrographs showing upper crypt GCs in fixed colonic tissue sections from ConvR, GF, and ConvD mice stained for Duox2 (red), mucus (UEA1; green), actin (grey), and DNA (blue). Intracellular Duox2 in GCs are indicated (yellow arrowheads). (J) Ex vivo mucus growth in Duox2fl/fl and Duox2ΔIEC colon tissue treated with carbachol (CCh), LPS, or P3CSK4. (K) Ex vivo mucus growth in WT colon tissue treated with P3CSK4 in the presence or absence of the Nox1 inhibitor ML171. Data represent n = 2–5 animals per group, as indicated. All data are pooled from at least two independent experiments or litters. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by DESeq2 (B, C, and E), Kruskal–Wallis and Dunn’s multiple comparison (F), or two-way ANOVA and Fisher’s LSD (J and K); P < 0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). Scale bars are 50 µm (H) or 5 µm (I). FC, fold change.

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