PCA and hierarchical clustering.

Principal component analysis of all Duodenum samples (A) and all Ileum samples (B). Correlation in gene expression and hierarchical clustering among all Duodenum samples (C) and all Ileum samples (D). . E. & F. Heat map of differentially expressed genes displayed with hierarchical clustering in all Duodenum samples (E) and in all Ileum samples (F). DE genes (time course using LRT) were defined as adjusted P-value <= 0.005.

Gene ontology enrichment analysis of selected gene expression clusters.

For each gene cluster, the expression pattern, number of genes present, and abundance score are indicated on the left. On the right, the top (top 30, except for cluster 5) overrepresented gene ontology terms in the cluster are indicated.

Expression of intestinal epithelial and mesenchymal markers.

Gene expression in the duodenum (black) and ileum (red) by normalized gene count across the first month of postnatal life. Highlighted genes include general markers of the intestine (A), intestinal epithelium and secretory populations (B), intestinal stem cells (C), enteroendocrine cells (D), and intestinal mesenchyme (E). Statistical analysis was by 2-way ANOVA compared to day 0. (F) qRT-PCR analysis of select genes from duplicate intestinal samples at the indicated time points. *p< 0.05, *duo – duodenum significant only, *ile- ileum significant only, ***p<0.001.

Expression of key epithelial genes in human tissues.

A. RNAScope of biopsied human tissue from the indicated gestational ages (Clinical data from the specimens is indicated in Table 2). DAPI is in blue, LGR5 in red. B. RNA expression in human intestinal organoids from 22-week fetus and 37-week infant by qRT-PCR. ** p<0.01.

Average Expression Duodenum and Ileum Defensins by RNASeq

Demographic data from human biopsy samples

Demographic data from human organoids

Expression of intestinal Wnts and differentiation factors.

Gene expression in the duodenum (black) and ileum (red) by normalized gene count across the first month of postnatal life. Highlighted genes include Wnt family members (A) and an array of downstream regulators and trophic factors known to be important for intestinal epithelial regulation (B). Statistical analysis was by 2-way ANOVA compared to day 0. (C) qRT-PCR analysis of Wnt3 from duplicate intestinal samples at the indicated time points. *p<0.05, *duo – duodenum significant only, *ile-ileum significant only. **p<0.01

Expression of immune genes.

A. Volcano plot of all differentially expressed genes from day 0 to week 4 in the duodenum and ileum. Highlighted genes are enriched for immune regulators. B. Gene expression of all alpha defensins in the duodenum and ileum by normalized gene count across the first month of postnatal life. Expression of adaptive immune cell markers (C) and innate lymphoid cell markers (D) by normalized gene count. (E) qRT-PCR analysis of Rora from duplicate intestinal samples at the indicated time points. (F) Expression of macrophage markers by normalized gene count. We used one-way ANOVA within each group and indicated significant values within the group as compared to day 0. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; *duo – duodenum only; *ile - ileum only

Protein expression of key markers in the ileum using immunofluorescence.

Ileal tissue was stained with E-cadherin (green) and DAPI (blue) as well as Lyz1 (grey, column 1), Olfm4 (red, column 2), MHCII (grey, column 3), and CD19 (magenta, column 4) at the indicated time points. Scale bar = 100 microns.

Extracellular adenosine metabolism genes are differentially expressed in immature versus mature ileum.

(A) Schematic of extracellular adenosine signaling. (B) Gene expression in the ileum of critical components of the extracellular adenosine pathway by normalized gene count across the first month of postnatal life in mice. (C) qRT-PCR analysis of select genes from duplicate intestinal samples at the indicated time points. (D) 10X magnification images of human epithelial organoids from the indicated age donors (Clinical data from the specimens is indicated in Table 2). (E) Gene expression in the human enteroids by qRT-PCR. Statistical analysis was by 2-way ANOVA compared to day 0. *p<0.05; **p<0.01; ***p<0.001