Postnatal maturation of the colonic IML barrier. (A) Sampling time points in postnatal days (P) of neonatal, weaned, and adult Wistar rats. (B) IML thickness quantified by ex vivo needle measurement. (C) IML barrier function quantified by ex vivo microbead penetration. (D) Representative confocal z-stacks used to generate data shown in C. Images show x/z-axis cross-sections of colonic tissues overlaid with microbeads. Impenetrable mucus is indicated (yellow arrows). (E) Confocal micrographs of fixed colonic tissue sections stained for DNA (grey) and Muc2 (green). Dashed lines in high-magnification P2 and P9 images show tissue surface (orange) and microbiota border (blue). (F) IML growth rate quantified by ex vivo needle measurement. (G) Principal component analysis plot of complete mucus proteome data. Samples are color coded based on age-dependent IML ex vivo phenotype: P1–P2 (low barrier function, slow growth; pink), P3–P7 (high barrier function, slow growth; teal), and P9–Adult (high barrier function, fast growth; red). (H) Volcano plot comparing complete mucus proteome data from slow growth (P1–P7) and fast growth (P9–Adult) samples. Proteins significantly enriched in slow (teal) or fast (red) growth groups are indicated. (I) Abundance of Muc2 (left), Tgm3 (middle), and Clca1 (right) proteins at different ages from label-free quantification (LFQ) of complete mucus proteome data. LOWESS curves (purple) are indicated for each dataset. Spearman correlation R and P values derived from correlating age and protein LFQ are shown. (J) Correlation of ex vivo mucus growth rate and Clca1 mucus proteome abundance (LFQ) at different ages. Simple linear regression (purple) is indicated. Pearson correlation R and P value derived from correlating mucus growth rate and Clca1 LFQ are shown. (K) Ex vivo mucus growth and response to metalloprotease inhibition by EDTA (left) or Comp PIC (right) treatment at different ages. Graphs show changes in mucus growth rate (Δ mucus growth; right) in response to inhibitor treatment. (L) AB/PAS-stained distal colon tissue sections from animals at different ages. Yellow dashed line indicates the IML. (M) Quantification of IML thickness values at different ages based on images shown in L. Data represent n = 5–19 (A–J, L, and M) or n = 5 (K) animals per group, as indicated. All data are pooled from at least three independent litters. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by Kruskal–Wallis and Dunn’s multiple comparison (B, C, F, K, and M) or Welch’s t test and Benjamini–Hochberg FDR correction (H); P < 0.05 (*), <0.01 (**). Image scale bars are 100 µm. AUC, area under the curve.
Figure 1.

Postnatal maturation of the colonic IML barrier. (A) Sampling time points in postnatal days (P) of neonatal, weaned, and adult Wistar rats. (B) IML thickness quantified by ex vivo needle measurement. (C) IML barrier function quantified by ex vivo microbead penetration. (D) Representative confocal z-stacks used to generate data shown in C. Images show x/z-axis cross-sections of colonic tissues overlaid with microbeads. Impenetrable mucus is indicated (yellow arrows). (E) Confocal micrographs of fixed colonic tissue sections stained for DNA (grey) and Muc2 (green). Dashed lines in high-magnification P2 and P9 images show tissue surface (orange) and microbiota border (blue). (F) IML growth rate quantified by ex vivo needle measurement. (G) Principal component analysis plot of complete mucus proteome data. Samples are color coded based on age-dependent IML ex vivo phenotype: P1–P2 (low barrier function, slow growth; pink), P3–P7 (high barrier function, slow growth; teal), and P9–Adult (high barrier function, fast growth; red). (H) Volcano plot comparing complete mucus proteome data from slow growth (P1–P7) and fast growth (P9–Adult) samples. Proteins significantly enriched in slow (teal) or fast (red) growth groups are indicated. (I) Abundance of Muc2 (left), Tgm3 (middle), and Clca1 (right) proteins at different ages from label-free quantification (LFQ) of complete mucus proteome data. LOWESS curves (purple) are indicated for each dataset. Spearman correlation R and P values derived from correlating age and protein LFQ are shown. (J) Correlation of ex vivo mucus growth rate and Clca1 mucus proteome abundance (LFQ) at different ages. Simple linear regression (purple) is indicated. Pearson correlation R and P value derived from correlating mucus growth rate and Clca1 LFQ are shown. (K) Ex vivo mucus growth and response to metalloprotease inhibition by EDTA (left) or Comp PIC (right) treatment at different ages. Graphs show changes in mucus growth rate (Δ mucus growth; right) in response to inhibitor treatment. (L) AB/PAS-stained distal colon tissue sections from animals at different ages. Yellow dashed line indicates the IML. (M) Quantification of IML thickness values at different ages based on images shown in L. Data represent n = 5–19 (A–J, L, and M) or n = 5 (K) animals per group, as indicated. All data are pooled from at least three independent litters. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by Kruskal–Wallis and Dunn’s multiple comparison (B, C, F, K, and M) or Welch’s t test and Benjamini–Hochberg FDR correction (H); P < 0.05 (*), <0.01 (**). Image scale bars are 100 µm. AUC, area under the curve.

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