662 - Temporal analysis of postnatal terminal ileum development and maturation by single cell transcriptomics

Publication Number: 1639.662

Aparna Venkatraman, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Shiyuan Chen, NA, Kansas City, MO, United States; Sherry M. Mabry, Children's Mercy, Kansas City, MO, United States; Daniel Louiselle, Children's Mercy Kansas City, Kansas City, MO, United States

Background: The terminal ileum plays a critical role in microbial sensing, nutrient absorption, barrier integrity, and immune coordination, all orchestrated by a single-layered epithelium. Despite its importance, the postnatal maturation of this region remains poorly characterized. Disruption of epithelial and innate immune programs contributes to neonatal intestinal diseases such as necrotizing enterocolitis (NEC). We previously identified loss-of-function variants in the single immunoglobulin interleukin-1–related receptor (SIGIRR), a negative regulator of Toll-like receptor signaling,in preterm infants with NEC, implicating dysregulated epithelial innate immunity. Understanding cell type specific developmental transcriptional programs is critical to reveal mechanisms of normal ileal maturation and disease susceptibility.

Objective: To map molecular and cellular programs underlying terminal ileum development using single-cell RNA sequencing (scRNA-seq) across postnatal days (P) 8, 14, and 25, and to determine how the NEC-associated Sigirrmu/mu (p.Y168X) mutation affects these programs at P8 in mice.

Design/Methods: Tissues were dissociated and processed using the 10x Genomics platform, with data analyzed via Cell Ranger and Seurat for alignment, clustering, and annotation. Pathway enrichment analysis and differentially expressed genes revealed stage-specific transcriptional programs.

Results: We identified 33 transcriptionally distinct cell types in the developing ileum. By P25, epithelial maturation was marked by enhanced mitochondrial respiration, lipid/cholesterol metabolism, and ER biosynthesis. Villus enterocytes upregulated DUOXA2, DUOX2, REG3, REG3B, SLC28A3, PLA2G5, and CAR4, reflecting antimicrobial defense and absorptive specialization. Crypt cells exhibited increased stemness, immune readiness, and metabolic activity regulated by SOX2, KLF4, TCF3, and FOXM1, while downregulating receptor signaling and Polycomb-repressed developmental genes. In Sigirrmu/mu mutants, epithelial maturation was impaired, with loss of extracellular matrix and adhesion pathways, reduced goblet cell effectors (AGR2, FCGBP), disrupted mucin/O-glycan biosynthesis, and decreased NKX2-3 expression. These defects compromised epithelial structure, secretory function, and immune coordination.

Conclusion(s): Overall, postnatal ileal development transitions from a proliferative to a metabolically active, immune-primed state and mature barrier integrity. SIGIRR is essential for this maturation and its loss reveals how innate immune dysregulation contributes to neonatal intestinal disease.

Figure 1 Single cell identifies cell types in the developing terminal ileum.
Figure1.pdfSchematic
illustration of experimental design, A. Single cell atlas annotated by cell type, B. integrated
UMAP projection of cell type cluster following assignment of cell type, C. Dot plots of relative
expression and percentage of cells expressing marker genes,D-F. UMAP projection of cell type
cluster following assignment of cell type for different time points, G. Quantitative of percentages
of major population in different time points, H. Heat map of relative expression of subsets of
epithelium, mesenchyme and immune cell, I-K.


Figure2 Differential gene expression in intestinal crypts and villi during postnatal development.
Figure 2.pdfVolcano plots showing differentially expressed genes (DEGs) in the crypt of P25 compared with to P8 (A) and P14 (D). Heatmaps showing DEGs in the crypt of P25 compared with P8 (B) and P14(E). Bar plots of differentially regulated transcription factors (TFs) in crypt of P25 compared to P8 (C) and P14 (F). Volcano plot showing DEGs in villus of P25 compared with P8 (G) and P14 (H). Dot plots showing upregulated and downregulated TFs in P25 compared with P8 (I) P14 (J). Dot plots of upregulated and downregulated TFs of P25 compared with P8 (K). Bar plots showing differentially regulated TFs in villus of P25 compared to P14 (L). Immuno-fluorescence staining of the terminal ileum at different postnatal developmental stages (M).


Figure 3. Differential gene expression in intestinal crypts and villi of Sigirr mutant and wild-type mice at P8.
Figure 3.pdfVolcano plots showing differentially expressed genes (DEGs) in the crypt (A) and villus (B)
of Sigirr mutant mice compared with wild-type (WT) littermates at P8. Dot plots showing the top-ranked DEGs in the crypt of Sigirr mutant mice compared with WT, (C). Gene Ontology (GO) term analysis of downregulated genes in the crypts of Sigirr mutant mice compared with WT, (D). Downregulation of lineage-specific cell genes in villus (E) and transcription factors (F) in Sigirr mutant crypts compared with WT. GO term analysis of DEGs in villus cells of Sigirr mutant mice compared with WT, (G).