Extended Data Fig. 8: Ptger4 ablation does not affect epithelial lineage differentiation and stem-cell function.

a, Ptger4 gene expression in crypts isolated from the ileum of littermate Ptger4^f/f and Ptger4^ΔIEC mice (n = 3 mice per genotype) and in organoids grown from these crypts (n = 3 cultures per genotype) determined by RT–qPCR analysis. Two-tailed unpaired t-test. b, Single-cell RNA-seq (Drop-seq) was performed in crypt epithelial cells isolated from littermate Ptger4^f/f and Ptger4^ΔIEC mice. Data for 2,439 single epithelial cells are shown in a t-SNE plot. c, Biological replicates visualized on a t-SNE plot. Crypt epithelial cells were independently isolated from two groups of mice per genotype (biological replicates 1 and 2). From the first biological replicate, two independent Drop-seq samples were collected (A and B) for a total number of three samples per genotype. d, Clustering and cluster assignments of 2,439 single epithelial cells displayed on a t-SNE plot. e, Proportion of each epithelial cluster among total crypt epithelial cells in Ptger4^f/f and Ptger4^ΔIEC mice. f, Violin plots showing the entire range of expression levels for a metagene of the β-catenin transcriptional program in n = 2,439 single epithelial cells from Ptger4^f/f and Ptger4^ΔIEC mice. g, Analysis of all Ptger4-expressing single cells detected (n = 478). Re-clustering results of Ptger4-expressing single cells with cluster annotations are visualized on a t-SNE plot. The expression levels of key marker genes for these clusters are visualized on t-SNE plots. h, Lineage tracing of Ptger4 heterozygous (Ptger4-HET) and Ptger4-knockout (Ptger4-KO) Lgr5⁺ stem cells. The small intestines of Lgr5-creERT2-Rosa26^tdTomato/+Ptger4^f/+ (Ptger4-HET) and Lgr5-creERT2-Rosa26^tdTomato/+Ptger4^f/f (Ptger4-KO) mice were examined for direct tdTomato fluorescence 5 days after a single injection of 2 mg tamoxifen per mouse. The results shown are representative of independent observations from one experiment. Scale bars, 70 μm. i, Quantification of intestinal epithelial populations in the ileum of littermate Ptger4^f/f (n = 5) and Ptger4^ΔIEC (n = 5) mice. Immunostaining was performed for markers of Paneth cells (lysozyme), tuft cells (Dclk1), enteroendocrine cells (chromogranin A) and stem cells (Olfm4). Scale bars, 20 μm. Goblet cells were identified by PAS staining and enterocytes were detected by alkaline phosphatase enzymatic activity. Scale bars, 50 μm. Incorporation and immunohistochemical detection of BrdU was used to determine the numbers of cycling cells. Scale bars, 50 μm. Data for each mouse represent mean number of positive cells per crypt or crypt–villus unit as indicated. n = 217–565 crypts and/or villi were evaluated per staining. Statistical comparisons were performed with two-tailed unpaired t-test except for PAS⁺ cells, for which unpaired Welch's t-test was applied. Mean ± s.e.m.; ns, non-significant; **P < 0.01.

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