Fig 2: RSPO3 controls secretory cell differentiation in corpus glands.(A) qPCR for expression of Rspo1–4 in Bl6 mice (n = 4). (B) ISH of Rspo3 (red) in corpus tissue of a nontreated MYH11-CreERT2; Rspo3WT/WT control mouse. (C) qPCR for Rspo3 expression in Rspo3-KO mice (n = 3) versus littermate controls (n = 4) and MYH11CreERT2-Rosa26CagRspo3 (Rspo3 KI) (n = 6) versus littermate controls (n = 6). (D) Immunofluorescence labeling for Ki67 (white) representative of Rspo3-KO and Rspo3-KI mice and littermate controls. (E) Location and relative size of the Ki67-positive gland compartment. (F) Quantification of the number of Ki67+ nuclei per gland in Rspo3-KO and Rspo3-KI mice and corresponding littermate controls (n = 3 mice per group). (G) Immunofluorescence labeling for GIF (green) representative of Rspo3-KO and Rspo3-KI mice and littermate controls. (H) Location and relative size of GIF-positive gland compartment. (I) Quantification of the number of GIF+ cells per gland in Rspo3-KO and Rspo3-KI mice and corresponding littermate controls (n = 3 mice per group). (J) Immunofluorescence labeling for GSII (red) representative of Rspo3-KO and Rspo3-KI mice and littermate controls. (K) Location and relative size of the GSII-positive gland compartment. (L) Quantification of the number of GSII+ cells per gland in Rspo3-KO and Rspo3-KI mice and corresponding littermate controls (n = 3 mice per group). (M) Immunofluorescence images of H/K-ATPase labeling (gray) in sections representative of Rspo3-KO and Rspo3-KI mice and corresponding littermate controls. (N) Quantification of parietal cells per gland in Rspo3-KO and Rspo3-KI mice and corresponding littermate controls (n = 3 mice per group). Mice were treated with tamoxifen 2 weeks before euthanasia. Scale bars: 100 μm. Enlargements in M equal 8:1 magnification. Unpaired parametric t test (C, E, F, H, and K); 1-way ANOVA with Tukey’s multiple-comparison test (A, I, and L).

Fig 3: Recapitulating Cycles of Colonic Epithelial Injury Regeneration In Vitro, Related to Figure 4(A) Expression of regenerative epithelial markers was analyzed by qPCR on monolayers re-submerged for 0hr, 4hrs, 8hrs, 24hrs and 7 days. Results were demonstrated by the relative expression fold changes in comparison to the 0hr time point. Student’s t test was performed to compare each individual time points after re-submersion to the 0hr time point. n = 3 samples/group.(B) Heatmap panel showing goblet cell, enteroendocrine cell, and colonocyte marker expression in monolayers re-submerged for the indicated time periods. Expression data are row normalized. (n = 3 replicates/time point).(C) ALI monolayers derived from HopxCreER/RosaTd mice were transiently labeled with 4OH-Tamoxifen 1 day before re-submersion on ALId21. Labeled Hopx+ cells were traced after re-submersion for 1 day and 7 days.(D) ALI monolayers subjected to 2 cycles of re-submersion and re-ALI stained with H&E staining, and for the indicated biomarkers of major cell lineages (Chga for enteroendocrine and UEA1 for goblet cell), and proliferating cells (Ki67).Mean values ± SD are shown. Two-tailed Student's t test: ∗p < 0.05; ∗∗p < 0.01. Bars: (C and D) 25 μm. Histological and immuno-fluorescent images are representative of at least three samples examined.

Fig 4: In Vitro Modeling of Hopx+ CARSCs(A and B) Whole mount images of ALId0 monolayers from WT, Lgr5GFP, and HopxGFP mice assessed for GFP (green) expression and co-stained for β-catenin (red) (A). Percentage of GFP+ cells in these monolayers as examined by flow cytometry (B).(C) Whole mount images of ALId0 monolayers stained for fetal markers Ly6a, Tacstd2, and Ly6g.(D) Heatmap of selected fetal markers in cells analyzed on ALId0, ALId4, ALId7, ALId14, and ALId21. Data are row normalized. n = 2 replicates/time point.(E and F) GSEA-based analysis comparing the mRNA profiles of ALId0 cells to the regenerative epithelium of DSS-treated mice (E) and fetal spheroid epithelium (F) (Mustata et al., 2013, Yui et al., 2018). Normalized enrichment score (NES) = 2.03 (E) and 2.33 (F). False discovery rate (FDR) < 0.001. See also Tables S3 and S4.(G) Pathway analysis (Enrichr) comparing the transcriptional signature of ALId1 cells to ALId0 cells.(H and I) Whole mount images for Ki67 staining (green) of HopxCreER/RosaTd-labeled cells (red) at early ALI time points (H). The proliferative rate of HopxCreER-labeled cells at designated ALI time points was plotted as mean ± SD (I). n = 3 samples/group.(J) Co-staining of Ki67 (green) and UEA1 (gray, goblet cells) with respect to Hopx+ clones (red) lineage-traced from ALId0 to ALId21.(K) A diagram showing that the in vitro Hopx+ CARSCs are highly proliferative on ALId0 to ALId2 and are capable of regenerating and giving rise to a homeostasis-like mature ALI monolayer.Two-tailed Student’s t test: ∗p < 0.05; ∗∗p < 0.01. Bars: (A, C, H, and J) 50 μm. Histological, immuno-fluorescent, and flow cytometry images are representative of at least 3 Transwell samples examined.See also Figure S3.

Fig 5: ADK-L modulates TBI-induced cell proliferation. (A) Immunofluorescence of the cell proliferation marker Ki67 in Adk-Ltg and AdkΔneuron mice compared with respective WT littermate controls at 3 days post injury; a magnified image of proliferative cells is shown in the close-ups. (B) Relative number of Ki67-positive cells normalized to WT. (C) Relative number of cells double labelled for Ki67 and DCX normalized to WT. (D) Confocal images with orthogonal projections illustrating the identity of ADK-positive cells in AdkΔneuron mice (DCX-positive cells co-express ADK (top) and GFAP-positive cells co-express ADK (bottom)). Scale bar is 200 µm in A and 50 µm in D. All values are presented as mean ± SEM (n = 5–9 mice per genotype). Two-tailed unpaired Student t-tests were used in B and C. * P ≤ 0.05, ** P ≤ 0.01, ** *= P ≤ 0.001, NS = no significance.