Microbiota colonization and GC maturation. (A) Sampling time points in postnatal days (P) of neonatal, weaned, and adult ConvR and GF mice. (B) Illustration of postnatal gene expression patterns with positive or negative monotonic correlation to age. (C) Total number of GC-enriched genes with positive or negative monotonic correlation to age in both ConvR and GF mice (microbiota independent) or in only ConvR or GF mice (microbiota dependent) was determined by Spearman correlation analysis of DESeq2 normalized RNA sequencing data. (D) Proportion of microbiota-dependent gene expression patterns shown in C identified by standalone DESeq2 comparison of ConvR and GF samples at individual ages. Genes not identified in any pairwise comparison are classified as “not identified” (NID). (E) Comparison of Spearman correlation coefficients (R) and GC expression enrichment (log₂ GC:enterocyte [EC] expression ratios) of genes with significant positive (orange) or negative (blue) monotonic correlation to age. Plots show GC-enriched genes with microbiota-independent (left) or -dependent (middle, right) expression patterns. (F) Heatmap showing standardized expression (z-score) of selected GC-enriched genes with significant microbiota-dependent or -independent expression correlation to age. (G) Confocal micrographs of fixed colonic tissue from adult ConvR and GF mice stained for DNA (grey) and Apo-Muc2 (red). (H) Quantification of Apo-Muc2–positive cells as a fraction of total epithelial cells in adult ConvR and GF tissue based on images shown in G. (I) Quantification Apo-Muc2–positive cells as a fraction of total epithelial cells in P1–9 neonatal ConvR and GF tissues based on images shown in Fig. S1 C. (J) Whole-mount confocal imaging of neonatal and adult RedMUC2^98tr colon stained for DNA (blue), F-actin (green), and MUC2-mCherry (red). Crypt entrances (yellow line) and intercrypt transition zones between high and low GC density areas (white dashed line) are indicated. Images show x/y-axis maximum intensity projections. Data represent n = 2–4 (C–F), n = 4–7 (G–I), or n = 4 (J) 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 Spearman correlation with Benjamini–Hochberg FDR correction (C–F), Mann–Whitney test (H), or two-way ANOVA and Fisher’s Least Significant Difference (LSD) (I); P < <0.001 (***), <0.0001 (****). Image scale bars are 50 µm.