Figure 2. ieCtnnb1 knockout altered cellular composition and expression profiles of small intestinal crypts.

(A) Schematic illustration of single-cell sequencing. Crypts were extracted from small intestines followed by fluorescence activated cell sorting (FACS) to enrich EpCAM+ DAPI- epithelial cells. Cells of two 10-week-old female mice for each genotype were pooled together to perform single-cell transcriptome analyses. (B) Uniform Manifold Approximation and Projection (UMAP) were used to visualize the clustering of 11,824 single cells from H11^i.enh mice and 8,094 single cells from Ctnnb1^Δi.enh mice. Cell types were assigned according to expressions of marker genes. CBC, crypt base columnar cell; TAC, transit-amplifying cell; EEC, enteroendocrine cell; imEC, immature enterocytes; mEC, mature enterocytes; GC, goblet cell; PC, Paneth cell; TC, tuft cell. (C) Expression and distribution of Ctnnb1 in small intestinal crypt cells of H11^i.enh and Ctnnb1^Δi.enh mice. (D) Violin plots showing the expression of Ctnnb1 in CBC, TAC, PC, GC, imEC, mEC; and the expression of Axin2 in CBC and TAC, of H11^i.enh and Ctnnb1^Δi.enh mice. (E) Comparison of the proportion of indicated small intestinal crypt cell types in H11^i.enh and Ctnnb1^Δi.enh mice. (F) Immunohistochemistry (left and middle) and quantification (right) of PCs in small intestines of H11^i.enh (n=6) and Ctnnb1^Δi.enh (n=6) mice. (G) Quantitative reverse transcription PCR (RT-qPCR) showing relative mRNA levels of PC marker genes (Lyz1, Pla2g2a, Wnt3, Math1) in small intestinal crypts of H11^i.enh (n=3) and Ctnnb1^Δi.enh (n=3) mice. (H) Distribution of G2M cells in H11^i.enh and Ctnnb1^Δi.enh small intestinal crypts, based on the expression of cell cycle marker gene Mki67. (I) Comparison of the proportion of G2M cells in CBC and TAC of H11^i.enh and Ctnnb1^Δi.enh small intestinal crypts. (J) Violin plots showing the expression of Mki67 in CBC and TAC of H11^i.enh and Ctnnb1^Δi.enh small intestinal crypts. (K) Immunohistochemistry (left and middle) and quantification (right) of Ki67+ cells in small intestines of H11^i.enh (n=6) and Ctnnb1^Δi.enh (n=6) mice. (L) Immunofluorescence (left and middle) and quantification (right) of EdU+ cells (red) in small intestines of H11^i.enh (n=6) and Ctnnb1^Δi.enh (n=6) mice after 4 hr EdU injection. Nuclei were labeled with DAPI (blue). (M–N) Violin plots showing expressions of marker genes for secretory cells (Lyz1, Ang4, Muc2, Mmp7) and absorptive cells (Fabp6, Apoa1, Fabp2, Reg3b) in secretory(M) and absorptive lineages (N) of H11^i.enh and Ctnnb1^Δi.enh small intestinal crypts. Scale bars, 50 μm (F, K, and L). Quantification data are shown as means ± SEM, statistical significance was determined using an unpaired two-tailed Student’s t-test (D, F, J, K, L, M, and N). Quantification data are shown as means ± SD, statistical significance was determined using Multiple t-tests – one per row (G). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns, not significant.

Figure 2—figure supplement 1. Single-cell survey of small intestinal crypt cells upon ieCtnnb1 knockout.

(A) Flow cytometry assays for isolating EpCAM+ DAPI- epithelial cells from H11^i.enh (n=2) and Ctnnb1^Δi.enh (n=2) small intestinal crypts. (B) Results of cell filtration using DoubletFinder. 1,294 doublets of 11,824 cells from H11^i.enh crypts and 567 doublets of 8,094 cells from Ctnnb1^Δi.enh crypts were identified (blue dots: singlets, red dots: doublets). (C) Results of cell filtration using Seurat. Cells with more than 30% of reads derived from mitochondrial genes are considered dead and removed from the analysis. (D) Cell-type-specific signatures. Heatmap showing relative expression levels (row-wise Z scores) of genes (rows) in cell-type-specific signatures. (E) Uniform Manifold Approximation and Projection (UMAP) visualizing LacZ+ single cells (black dots) of H11^i.enh small intestinal crypts (n=4 mice). (F) Alcian Blue PAS staining and quantification of goblet cells in the small intestine of H11^i.enh (n=6) and Ctnnb1^Δi.enh (n=6) mice. (G) Violin plots showing expressions of marker genes for secretory cells (Lyz1, Ang4, Muc2, Mmp7) and absorptive cells (Fabp6, Apoa1, Fabp2, Reg3b) in H11^i.enh and Ctnnb1^Δi.enh crypt base columnar (CBCs) and transit-amplifying cells (TACs). (H–J) Gene ontology (GO) analysis of differential genes in stem cell lineage (H), secretory lineage (I), and mature enterocytes (J) of H11^i.enh and Ctnnb1^Δi.enh crypts. Scale bars, 50 μm (E), 10 μm (magnified views in E). Quantification data are shown as means ± SEM, statistical significance was determined using an unpaired two-tailed Student’s t-test (F and G). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns, not significant.