Figure 4. 1PAT effects on ileal gene expression from P2 to P23, based on RNA-Seq analysis.

(A) Bray-Curtis analysis of ileal gene expression of pups at P2 and P12 (2 days after PAT), represented by PCA. All differences between expression at P2 and P12 (in both 1PAT and control mice) were significant in both males and females (p<0.05). (B) Differential ileal gene expression between P2 males and females; volcano plot indicates 1514 genes with significantly different expression. DESeq2 difference visualization maximizes at 15 (-log₁₀ (adjusted p values)). (C) Ileal KEGG pathways altered by 1PAT in P12 males; 23 significant pathways (adjusted p<0.05, after Benjamini-Hochberg correction), bars indicate numbers of differentially affected genes/pathway. (D) Differential ileal gene expression in 1PAT and control P23 females (left) and males (right) shown by volcano plot. In females, 124 genes (55 up-, 69 down-), and in males, 1511 genes (518 up-, 993 down-) were significantly differentially expressed, a 12.2-fold difference between the sexes. n = 4, 3, and 6–7 mice per group, at P2, P12, and P23, respectively. [See also Figure 4—figure supplements 1–3, and Supplementary files 3 and 4].

Figure 4—figure supplement 1. Differential ileal gene expression maturation between P2 and P12 in control and PAT mice, as shown in volcano plots.

In the controls, a total of 7920 genes (for males) and 6394 (for females) showed significant maturation between P2 and P12, (adjusted p<0.05 based on DESeq2 differential expression analysis). The PAT mice received their antibiotic course between P5-10, and the number of genes maturing between P2 and P12 fell to 6565 in males, 17% less than in controls, and to 5017 in females, 21% less than in controls. Visualization of DESeq2 differences maximizes at 15 (-log₁₀ (adjusted p values)).

Figure 4—figure supplement 2. Unsupervised hierarchical clustering of significantly differential genes in P23 1PAT and control mice as assessed by RNA-Seq, and shown on heat maps.

The number of differential genes differed substantially between females (left, 124 genes) and males (right, 1511). The differential gene lists can be found in Supplementary file 3.

Figure 4—figure supplement 3. Effect of 1PAT on ileal gene expression profiles in 1PAT and control male mice at P42, showing an unsupervised hierarchical clustering of all significantly differential genes.

817 genes showed significant differential expression (334 up-, 483 down-).