Unique Gene Expression Signatures in the Intestinal Mucosa and Organoids Derived from Germ-Free and Monoassociated Mice

Figures

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  Figure 1. The immune system is not reconstituted upon monoassociation with a single E. coli strain. (A) Venn diagrams indicating numbers of gene probes differentially expressed in the middle (SI-middle) and distal (SI-distal) parts of the small intestine and in the colon of GF, N, and O animals when compared with CR mice (significance criterion: q < 0.05; |log FC| ≥ 1). The corresponding annotated genes are listed in Supplementary Table S2. (B) Genes expressed differentially in the analyzed groups of mice were analyzed using ‘Gene Ontology Biological Processes’ (GO BPs) and ‘Cell Types/Mouse Gene Atlas’ (CT/MGA) categories in Enricher datasets. The results were sorted according to the combined score (CS; CS is computed by multiplying the log of p-value obtained from the Fisher’s exact test by the z-score of the deviation from the expected rank). A maximum of ten GO BPs and CT/MGA categories containing at least two differentially expressed genes and with CS ≥ 1 is shown; the coloring of the columns corresponds to the significance of the individual columns in the graph. The number of genes in the particular category is indicated by the number before the parenthesis. |log FC|, absolute value…

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  Figure 2. A limited number of genes are uniquely expressed in the GF small intestine. (A) Venn diagrams comparing gene expression profiles in small intestinal segments of CR, N, and O animals when contrasted with the gene expression profile obtained in GF mice. Eleven gene probes (representing 10 annotated genes) and 9 gene probes (representing 8 annotated genes) were differentially expressed in the SI-middle or SI-distal segment, respectively (selection criterion: |log FC| ≥ 1; p < 0.05 in at least two of three comparisons). The genes are listed in Supplementary Table S3. (B) Validation of cDNA microarray by qRT-PCR. Diagrams depict expression analysis of the indicated genes performed by cDNA microarray hybridization (left) or by qRT-PCR (right). Normalized fluorescence signal obtained in the respective gene probe upon hybridization with RNA isolated from GF mice was set to 1. Results of qRT-PCR were normalized to ubiquitin B (Ubb); the expression level of the respective gene in the mucosa of GF mice was set to 1. Four mice from each group were analyzed. Error bars represent SDs. Corresponding CT values are listed in Supplementary Table S4; * p < 0.05; ** p < 0.01 [one-way Analysis of…

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  Figure 3. Aldh1a1 represents a robust marker of the GF colon. (A) A Venn diagram comparing gene expression profiles of the colonic mucosa of CR, N, and O animals when contrasted with the gene expression profile obtained in GF mice. According to the selection criterion (|log FC| ≥ 1; p < 0.05), 17 gene probes representing 12 annotated genes were identified. The genes are listed in Supplementary Table S3. (B) Validation of cDNA microarray by qRT-PCR. Comparison of expression analysis of the indicated genes performed by cDNA microarray hybridization (left) or by qRT-PCR (right). Samples obtained from four mice from each group were analyzed, as described in Figure 2B. Corresponding CT values are listed in Supplementary Table S4; * p < 0.05; ** p < 0.01 (one-way ANOVA test). (C) Immunohistochemical detection showing increased production of Aldh1a1 in differentiated epithelial cells (black arrows) of the colon of GF mice. Representative images of specimens counterstained with hematoxylin are shown. Scale bar: 0.15 mm.

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  Figure 4. Oc2 expression was elevated in organoids derived from the gnotobiotic colon (A) A Venn diagram comparing gene expression profiles of colon organoids derived from GF, N, and O animals when compared to colon organoids derived from CR mice; 366 gene probes representing 285 annotated genes were identified. The genes are listed in Supplementary Table S6. (B) Enricher analysis of 138 genes upregulated in GF vs. CR colon organoids. The diagram shows top ten categories of the Jensen Compartment (J COMP; https://compartments.jensenlab.org/Search). (C) Validation of cDNA microarray by qRT-PCR. Diagrams depict expression analysis of the indicated genes in GF vs. CR colon organoids performed by cDNA microarray hybridization (left) or by qRT-PCR analysis (right). Normalized fluorescence signal obtained in the respective gene probe upon hybridization with RNA isolated from CR mice was set to 1. Results of the qRT-PCR analysis were normalized to Ubb; the expression level of the respective gene in the mucosa of CR mice was set to 1. Organoids derived from at four two mice were used; qRT-PCR reactions were run in technical triplicates. Error bars represent SDs. Corresponding CT values are listed…