uea1 fitc  (Vector Laboratories)

Journal: The Journal of Experimental Medicine

Article Title: Neonatal microbiota colonization primes maturation of goblet cell–mediated protection in the pre-weaning colon

doi: 10.1084/jem.20241591

Figure Lengend Snippet: 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 ) 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 and are shown for illustrative purposes only.

Article Snippet: Lastly, slides were washed with PBS and counterstained with a Hoechst-34580 DNA dye (5 μg/ml; Merck) in some cases supplemented with combinations of Ulex europaeus agglutinin I (UEA1) Atto 488–conjugated lectin (10 µg/ml; Merck), UEA1 DyLight647–conjugated lectin (10 µg/ml; Vectorlabs), or Wheat germ agglutinin (WGA) Alexa 555–conjugated lectin (10 µg/ml; Thermo Fisher Scientific) for 15 min.

Techniques: Ex Vivo, Activation Assay, Staining, Imaging, Expressing, In Silico, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Neonatal microbiota colonization primes maturation of goblet cell–mediated protection in the pre-weaning colon

doi: 10.1084/jem.20241591

Figure Lengend Snippet: 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 Duox2 fl/fl and Duox2 ΔIEC 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.

Article Snippet: Lastly, slides were washed with PBS and counterstained with a Hoechst-34580 DNA dye (5 μg/ml; Merck) in some cases supplemented with combinations of Ulex europaeus agglutinin I (UEA1) Atto 488–conjugated lectin (10 µg/ml; Merck), UEA1 DyLight647–conjugated lectin (10 µg/ml; Vectorlabs), or Wheat germ agglutinin (WGA) Alexa 555–conjugated lectin (10 µg/ml; Thermo Fisher Scientific) for 15 min.

Techniques: Activation Assay, Expressing, Isolation, RNA Sequencing, Comparison, Gene Expression, Quantitative RT-PCR, Staining, In Situ, Hybridization, Ex Vivo