CREPT deficiency decelerates the daily renewal of intestinal epithelium

To study the role of CREPT in intestine maintenance, we deleted CREPT specifically in the intestinal epithelium (assigned Vil-CREPT^KO) by introducing villin-cre³³ into CREPT^fl/fl mice (Supplementary Fig. 1a). A Western blot demonstrated that CREPT was effectively deleted in the small and large intestines (Supplementary Fig. 1b). The low level of CREPT protein as detected by the Western blot might be due to the expression in non-epithelial cells in the intestine. A histological analysis showed that CREPT was completely deleted in the epithelial cells of small (Fig. 1a) and large intestines (Supplementary Fig. 1c) but remained observable levels in the connective tissue cells of Vil-CREPT^KO mice. Of note, CREPT protein is mainly expressed in the nuclei of crypt cells of wild type (WT) mice (Fig. 1a). A qRT-PCR analysis confirmed that the expression of CREPT is abundant in the crypts, similar to that of Olfm4, a marker of crypts (Fig. 1b).

a Representative images of fluorescent CREPT staining in small intestines from WT and Vil-CREPT^KO mice. Images are representative of n = 4 mice per genotype. b Quantitative RT-PCR analysis of CREPT and Olfm4 in crypt cells and epithelial cells (crypt + villous cells). p = 0.0067 (CREPT); p = 0.0002 (Olfm4). n = 3 independent experiments. c Representative images of WT and Vil-CREPT^KO mice. d Quantification of body weights of WT and Vil-CREPT^KO mice. n = 11 WT mice and n = 9 Vil-CREPT^KO mice. p < 0.0001. e Representative images of Ki67 staining in duodenums of WT and Vil-CREPT^KO mice. Images are representative of n = 4 mice per genotype. f Representative confocal images of cell migration in WT and Vil-CREPT^KO mouse at 2-h, 24-h, and 72-h after EdU injection. Images are representative of 3 independent experiments (n = 6 mice per genotype). g Quantification of EdU⁺ cell number in each crypt 2 h after EdU labeling. n = 3 independent experiments. p = 0.0435. h Quantification of EdU⁺ cells migration length at 24 h. The migration distance of the fastest cells was measured from the bottom of the crypts. n = 3 independent experiments. p = 0.0017. i Quantification of Edu⁺ cell migration length at 72 h. The migration distance of the slowest cells was measured from the bottom of the crypts. n = 3 independent experiments. p = 0.0412. j Representative images of Paneth cells staining in duodenums of WT and Vil-CREPT^KO mice. n = 4 mice per genotype. k Representative images of goblet cells staining in the jejunum of WT and Vil-CREPT^KO mice. n = 3 mice per genotype. l Quantification of goblet cell number per villus. n = 3 mice per genotype. p = 0.0002. Statistics data represent mean ± SEM. All p values were generated by 2-tailed Student’s t-test. Scale bars: 20 μm (a and j), 100 μm (e, f, k).

Vil-CREPT^KO mice were not born at the expected Mendelian ratio (Supplementary Fig. 1d). The survived Vil-CREPT^KO adult mice showed substantially decreased body size (Fig. 1c) and significant body weight loss (Fig. 1d). IHC analyses showed that the villus number and structure in the small intestine were normal (Supplementary Fig. 1e, top panel), but, the crypts in the large intestine were enlarged and became thick (Supplementary Fig. 1e, bottom panel) in the Vil-CREPT^KO mice. To address whether CREPT affects the status of epithelial cells, we stained the cells with Ki67, a widely used marker for cell proliferation. The results showed that Ki67 positive cells distributed in the villi were substantially reduced in Vil-CREPT^KO mice while those cells in the crypts appeared no difference between WT and Vil-CREPT^KO mice (Fig. 1e). Interestingly, the number of Ki67 positive cells was decreased in the crypts in the large intestine of Vil-CREPT^KO mice in comparison with WT mice (Supplementary Fig. 1f). To address whether the deletion of CREPT influences cell proliferation, we pulse-labeled the proliferating cells with EdU (5-ethynyl-2´-deoxyuridine)³⁴. The results showed that EdU-labeled cells were restricted to the crypt compartment in intestines after 2 h incorporation in both WT and Vil-CREPT^KO mice (Fig. 1f, top panel). However, the number of EdU-labeled cells was significantly decreased in Vil-CREPT^KO mice (Fig. 1g), which corresponded with a substantial decrease of S/G2/M phase cell numbers in Vil-CREPT^KO mice (Supplementary Fig. 1g). Furthermore, we observed that EdU⁺ cells migrated slowly in Vil-CREPT^KO mice in comparison with WT mice at 24 h after EdU labeling (Fig. 1f, middle panel, and 1 h). The EdU⁺ cells migrated to the top of villi in WT mice but were not extruded on the crypts-villi axis in Vil-CREPT^KO mice at 72 h after labeling (Fig. 1f, bottom panel, and 1i). The EdU⁺ cells showed similar patterns in the distribution at the large intestines in both WT and Vil-CREPT^KO mice at 24 and 48 h after EdU labeling (Supplementary Fig. 1h). As for differentiated intestinal cells, the number of lysozyme stained Paneth cells (Fig. 1j) of small intestines and alcian blue stained goblet cells of small (Fig. 1k, l) and large (Supplementary Fig. 1i) intestines were significantly decreased in Vil-CREPT^KO mice. Taken together, these results suggest that the deletion of CREPT in epithelial cells leads to an abnormal crypt structure in the large intestine, slower migration of epithelial cells in villus in the small intestine, and lower number of proliferating cells in the small intestine.

CREPT deficiency in the epithelium decreases Lgr5⁺ ISCs and impairs organoid formation

Since CREPT deletion decelerated the turn-over rate of intestinal epithelial cells and decreased the number of proliferated and differentiated cells, we questioned if CREPT deletion might impair the function of ISCs. To determine whether a specific type of stem cell responds to CREPT deletion, we examined the putative ISC markers. The CBC stem cell markers (Lgr5 and Olfm4)^2,35 were downregulated in the crypts of Vil-CREPT^KO mice, whereas reserved stem cell markers (Dclk1 and Bmi1)^13,36, and both CBC and reversed ISC markers (Sox9 and Lrig1)^12,37 were barely affected (Fig. 2a). Indeed, the number of Lgr5-GFP⁺ stem cells was significantly decreased in Villin-cre; Lgr5-EGFP; CREPT^fl/fl mice (Fig. 2b, c, Supplementary Fig. 2a). The Lgr5-GFP mice are mosaic in adults with no GFP expression in a small part of crypts (Fig. 2b, upper panels). Since Villin-cre is expressed during gut development, it is possible that GFP-silenced patches might be preferentially advantaged due to CREPT deletion. To avoid this possibility, we examined the number of Lgr5-GFP⁺ cells in adult mice. Since CREPT was co-localized with Lgr5 and Ki67 (Fig. 2d and Supplementary Fig. 2b) and Lgr5-GFP⁺ cells had higher CREPT protein (Fig. 2e) and mRNA (Fig. 2f) level than Lgr5-GFP⁻ cells, we deleted CREPT in Lgr5⁺ cells of adult Lgr5-EGFP-ires-creERT2; CREPT^fl/fl (Lgr5-CREPT^KO) mice by addition of Tamoxifen (Supplementary Fig. 2c), and analyzed the Lgr5-GFP⁺ cells over time post Tamoxifen treatment (Fig. 2g). The results showed that the deletion of CREPT in Lgr5⁺ cells led to a declined number of Lgr5-GFP⁺ cells at 7 days post Tamoxifen treatment (Fig. 2h, i). On day 30, the number of Lgr5-GFP⁺ cells dropped to a quarter of that at day 0 (Fig. 2, i).

a A quantitative RT–PCR analysis of the expressions of CBC and reserve stem cell markers in WT and Vil-CREPT^KO crypt cells. b Representative images of Lgr5-GFP⁺ cells in WT and Vil-CREPT^KO intestines. c Quantification of Lgr5-GFP + cells in small intestines of WT and Vil-CREPT^KO mice based on FACS analyses. n = 3 independent experiments. p = 0.0042. d Representative images of Lgr5 and CREPT staining in intestine crypts. e A FACS analysis of Lgr5 and CREPT stained cell populations. The population of crypts cells, which had the highest level of Lgr5, also expressed the largest amount of CREPT protein. f A quantitative RT-PCR analysis of Lgr5 and CREPT in sorted Lgr5-GFP⁺ and Lgr5-GFP⁻ cells. n = 3 independent experiments. p < 0.0001 (Lgr5 in Lgr5⁻ vs. Lgr5⁺ cells). p = 0.0002 (CREPT in Lgr5⁻ vs. Lgr5⁺ cells). g A schematic diagram showing the TAM treatment in Lgr5-GFP; CREPT^fl/fl mice. h Representative images of Lgr5-GFP staining in intestines at indicated time post TAM treatment. i Percentage of Lgr5-GFP^high cells in crypts at indicated time post TAM treatment by FACS analyses. p = 0.0393 (day 1 vs. day 7). p = 0.0011 (day 1 vs. day 30). j Representative images of WT and CREPT deleted organoid formation derived from Lgr5-GFP and Lgr5-GFP; CREPT^fl/fl mice. k The numbers of WT and Lgr5-CREPT^KO organoids showing <100 μm and ≥100 μm diameters. Images are representative of at least three independent experiments. Statistics data represent mean ± SEM. All p values were generated by 2-tailed Student’s t-test. Scale bars: 100 μm (b, h, j), 10 μm (d).

To identify the role of CREPT in stem cells, ex vivo culture of intestinal organoids was generated from the intestinal crypts of WT and Vil-CREPT^KO mice. However, the CREPT deficient crypts failed to survive ex vivo (Supplementary Fig. 2d). To overcome the failure of organoid formation, we cultured organoids derived from Lgr5-GFP and Lgr5-GFP; CREPT^fl/fl mice, and then deleted CREPT by adding 4OH-Tamoxifen (4OHT) supplemented growth medium (Supplementary Fig. 2e). The results showed that the crypts from WT intestines budded at day 3 and formed organoids at day 5 (Supplementary Fig. 2f), however, the 4OH-TAM-induced CREPT deficient crypts produced fewer outgrowth at day 3 and failed to form mini guts at day 5 (Fig. 2j and Supplementary Fig. 2g). Quantification of 100 individual crypts showed CREPT deficient crypts had defective organoid-forming ability ex vivo (Fig. 2k). To evaluate the passage ability of organoids, we harvested the first generation of organoids from crypts and disaggregated organoids to form the second generation. The results showed that WT organoids were re-formed after another 5 days’ culture (Supplementary Fig. 2h), but, the Lgr5-CREPT^KO organoids failed to re-form after disaggregation (Supplementary Fig. 2i). Moreover, we verified the role of CREPT in human colorectal organoids. To deplete CREPT protein, we used a PROTAC, named PRTC, which was developed for specifically targeting CREPT and mediating its degradation³⁸, to treat human colorectal organoids. The result showed that fewer and smaller organoids formed after PRTC treatment (Supplementary Fig. 2j, k). A Western blot result showed that PRTC induced the decrease of CREPT protein in human organoids (Supplementary Fig. 2l). This result is consistent with the results of organoids formation from mouse crypt cells under CREPT deletion. All these results suggest that CREPT is essential for the maintenance of Lgr5⁺ ISCs and organoid formation.

The intestinal epithelium with CREPT deletion fails to regenerate after damage

The defects in the intestine and ISCs of Vil-CREPT^KO mice prompted us to examine whether CREPT plays a role in epithelial regeneration. We applied X-ray irradiation (10 Gy) and allowed the regeneration of intestinal villi and crypts for different times (Fig. 3a). The results showed that all Vil-CREPT^KO mice reached the euthanasia thresholds within 7 days, but 50% of WT mice remained survival after 14 days post-irradiation (dpi) (Fig. 3b). IHC analyses demonstrated that X-ray irradiation destructed the crypts, as showed by Olfm4⁺ staining, in small intestines of both WT and Vil-CREPT^KO mice from 0 to 3 dpi (Fig. 3c). At 5 dpi, the crypts (Olfm4⁺) and the villus recovered to normal structure in WT mice, however, the Olfm4⁺ crypts barely reformed and the villus structure remained disrupted in Vil-CREPT^KO mice (Fig. 3c, top panel). A quantitative analysis showed that the numbers of Olfm4⁺ crypts were significantly decreased in the Vil-CREPT^KO mice at 5 dpi (Fig. 3d). Simultaneously, we observed that crypts in the colon were destroyed at 3 dpi but recovered at 5 dpi in WT mice (Fig. 3c, bottom panel), however, the outgrowth of colorectal crypts of irradiated Vil-CREPT^KO mice was reduced significantly (Fig. 3c bottom panel and 3e). All these results suggest that the intestinal epithelial cells are unable to recover after irradiation when CREPT was deleted.

To validate the results in X-ray irradiation, we challenged the mice with DSS (dextran sodium sulfate), a chemical reagent that destroys the mouse colorectal epithelium and causes inflammation³⁹, for 7 days, and allowed epithelium recovery (Supplementary Fig. 3a). The results showed that no Vil-CREPT^KO mouse but 70% of WT mice survived for more than 20 days after DSS treatment (Supplementary Fig. 3b). The architecture of colorectal crypts remained disrupted in Vil-CREPT^KO mice but was completely recovered in WT mice on day 3 after DSS withdrawal (dpD) (Supplementary Fig. 3c). Taken together, all the results suggest that CREPT is critical for the regeneration of intestinal epithelial cells after destroyed by both physical and chemical reagents.

CREPT is required for the proliferation and differentiation of Lgr5⁺ stem cells

Next, we explored the function of CREPT in Lgr5⁺ stem cells by RNA sequencing (RNA-seq). We induced CREPT deletion in Lgr5-GFP⁺ cells by intraperitoneal injection of Tamoxifen into mice, and sorted the Lgr5-GFP^high cells from disaggregated WT and Lgr5-CREPT^KO intestines (Fig. 4a). RNA-seq results showed that 198 genes were downregulated by CREPT deletion at homeostasis (Fig. 4b). These genes include differentiation determining genes and cell cycle promoting genes (Fig. 4b, marked genes). Consistently, a Gene Ontology (GO) analysis highlighted that the downregulated genes in CREPT deleted Lgr5⁺ cells were enriched in the GO terms of cell differentiation and gene transcription regulation (Fig. 4c). Upregulated genes were those involved in apoptosis (Fig. 4d). In particular, three sets of lineage signature genes, Neurog3, Nkx2-2, Pdx1, and Pax6 (enteroendocrine cell), Spdef and Dll1 (goblet and Paneth cell), and Nox1 and Atoh1 (Paneth cell), were decreased in Lgr5-CREPT^KO cells (Fig. 4e). Interestingly, CREPT deletion caused downregulation of migration genes (Vim and Snai3), and increased the epithelium-related genes (Lama5 and collagen) (Fig. 4f). This gene expression alteration echoes the observation that EdU-labeled CREPT deleted cells migrated slower along the crypt-villi axis (see Fig. 1). In addition, the expression of cell cycle promoting genes in Lgr5-CREPT^KO cells were decreased (Fig. 4g) and EdU-labeled crypt numbers in Lgr5-CREPT^KO organoids were declined (Fig. 4h), suggesting that CREPT is crucial for the proliferation of Lgr5⁺ stem cells. Taken together, all the results suggest that CREPT maintains the proliferation and differentiation of Lgr5⁺ ISCs at homeostasis.

CREPT deficiency suppresses the expression of stem cell signature genes in crypts by repressing the proliferation of Lgr5⁺ ISCs

The RNA-seq data of TAM treated Lgr5+ cells showed that the expression of Lgr5⁺ ISC signature genes¹⁶, including the Lgr5 gene per se, was barely affected by CREPT deletion in purified Lgr5+ cells (Supplementary Fig. 4a). However, as mentioned above, we observed a significant decrease of Lgr5-GFP+ cells after TAM-induced CREPT deletion (Fig. 2h, i). It is possible that the cells with ISC signature genes were survived but other cells were lost in CREPT deleted crypts. The RNA-seq data of Lgr5+ cells only reflected the gene expression change in remaining Lgr5+ cells. To clarify the role of CREPT in ISC maintenance, we examined the gene expression in the intestinal crypts from WT and Vil-CREPTKO mice. An RNA-seq result showed that 412 genes were substantially downregulated in the crypts when CREPT was deleted (Supplementary Fig. 4b). We compared our RNA-seq data with a previously identified signature gene set of Lgr5⁺ stem cells¹⁶. The results showed that the ISC signature genes were enriched in the downregulated genes in Vil-CREPT^KO mice, suggesting that CREPT may affect the stem cells in crypts (Supplementary Fig. 4c). For validation, Lgr5 and Olfm4, two stem cell markers, were shown to be decreased in the crypts of Vil-CREPT^KO mice (Supplementary Fig. 4d). Simultaneously, we observed that cell cycle promoting genes and differentiation determining genes were downregulated (Supplementary Fig. 4e–g). Of note, Nox1 and Atoh1, two marker genes of Paneth cells, were substantially decreased in the crypts of Vil-CREPT^KO mice (Supplementary Fig. 4f, g), which echoes our immunostaining results for decreased lysozyme positive Paneth cells (Fig. 1j). Taken together, we conclude that CREPT participates in the regulation of genes in maintaining the proliferation of Lgr5⁺ ISCs.

CREPT is required for Wnt activation during the regeneration process

Next, we analyzed the gene expression profiles of irradiated WT and Vil-CREPT^KO intestines. Surprisingly, 993 genes were downregulated in irradiated Vil-CREPT^KO intestines (Fig. 5a), much more than that of untreated WT and Vil-CREPT^KO intestines (412 genes, Supplementary Fig. 4b). These results suggest that CREPT plays a more prominent role in regeneration rather than homeostasis maintenance. To examine the role of CREPT in intestine regeneration, the RNA-seq data of un-irradiated WT and irradiated WT intestines were analyzed (Supplementary Fig. 5a). A KEGG analysis to identify enriched pathways in intestinal cells after irradiation showed that PI3K-AKT, Hippo, Wnt, MAPK, and TGFβ signaling pathways were upregulated in irradiated WT intestines (Supplementary Fig. 5b). We hypothesized that CREPT deficiency might impede the activation of one or more of these five signaling pathways. Indeed, a gene-set enrichment analysis (GSEA)^40,41 of the gene expression profiles from irradiated WT and Vil-CREPT^KO intestines showed substantial upregulation and downregulation in Wnt signaling components (Supplementary Fig. 5c), and upregulation mostly in other four signaling pathways (Supplementary Fig. 5d–g). Therefore, we focus on the effect of CREPT on the Wnt signaling pathway.

a Volcano plot showing the upregulated and downregulated genes in irradiated (IR)-Vil-CREPT^KO crypts compared to IR-Vil-CREPT^WT ones. b Quantitative RT-PCR analysis of Wnt target genes in irradiated WT and Vil-CREPT^KO intestines at 3 dpi. n = 3 independent experiments. c Heatmap showing the expression levels of Wnt target genes in untreated/irradiated WT and Vil-CREPT^KO intestines. d Quantitative RT-PCR analysis of Cd44, Ascl2, EphB3, and Sox9 expression in untreated/irradiated WT and Vil-CREPT^KO intestines. p < 0.0001 by 2-tailed Student’s t-test. n = 3 independent experiments. e ChIP-seq signals of CREPT occupied genomic loci. The CREPT occupancy frequency is illustrated at the genome of Wnt target genes including Cd44, EphB1, Alcam, and Rspo2. f Quantitative PCRs showing the enrichment of CREPT on the promotor and termination region of the Cd44 gene. The primers A, B, and C, were designed as indicated. Images are representative of three independent experiments. Statistics data represent mean ± SEM.

Indeed, CREPT deletion caused substantial downregulation of a majority of Wnt target genes, although Myc, Ccnd1, EphB2, and Axin2 were unchanged or slightly unexpectedly upregulated (Fig. 5b). We then excluded the Wnt related genes with invariant and/or reduced expression value in irradiated WT mice compared to the untreated WT mice, and analyzed the expression of the increased Wnt responsible genes in the intestines of irradiated WT and Vil-CREPT^KO mice. The results showed that many Wnt positive-regulation genes were activated in irradiated WT mice (Fig. 5c, panel 1 vs. 3), but the upregulation was suppressed in irradiated Vil-CREPT^KO mice (Fig. 5c, panel 3 vs. 4). Quantitative RT-PCR analysis confirmed that CD44, Ascl2, EphB3, and Sox9, four Wnt targeted genes, were substantially increased after X-ray irradiation in WT mice but deletion of CREPT significantly impaired their expression (Fig. 5d). These results suggest that CREPT is essential for the Wnt signaling activation during the regeneration of intestinal epithelium.

To confirm the regulation of CREPT on Wnt target genes, we performed a ChIP-sequencing (ChIP-seq) experiment in WT and Vil-CREPT^KO intestinal crypts. The results demonstrated that CREPT occupancy was enriched at both the promoters and termination regions of Cd44 and EphB1, but only at the termination region of Alcam and at the promoter of Rspo2 (Fig. 5e). The ChIP-seq result was confirmed using the Cd44 gene by a ChIP experiment (Fig. 5f). These results suggest that CREPT regulates Wnt target genes at the transcriptional level but in different ways. To further confirm the role of CREPT on the regulation of Wnt signal, we used intestinal organoids in the presence of CHIR99021, a GSK3β inhibitor to impair the degradation of β-catenin in the cytoplasm⁴². The results showed that CHIR99021 substantially increased the Lgr5⁺ staining cells in WT organoids (Supplementary Fig. 5h, comparing β and δ, ϕ and η), but failed to maintain the organoid passage and Lgr5 expression in the Lgr5-CREPT^KO organoids (Supplementary Fig. 5h, i, comparing π and η). A quantitative analysis demonstrated substantial decreases in organoid numbers in CREPT KO cells in the presence of CHIR99021 (Supplementary Fig. 5j). These results suggest that CREPT is also essential for the Wnt-activated growth of intestinal organoids.

CREPT facilitates β-catenin retaining in the crypt nucleus during regeneration

The impacts of CREPT on the Wnt signaling during intestinal renewal and regeneration prompted us to explore how CREPT regulates Wnt signaling. To this end, we performed luciferase assays with a widely used super-top reporter⁴³. The results showed that the deletion of CREPT impaired Wnt, APC-depletion and β-catenin activated transcription of the reporter (Fig. 6a–c) but CHIR99021 greatly rescued the luciferase activity (Fig. 6d), suggesting that CREPT regulates the downstream events of Wnt signaling at the transcription level. However, the mRNA levels of β-catenin were barely affected by CREPT deficiency (Supplementary Fig. 6). Based on our previous studies^23,28,29, we speculate that CREPT regulates Wnt signaling through β-catenin interaction. Indeed, we observed that endogenous (Fig. 6e) and exogenous (Fig. 6f) CREPT and β-catenin proteins interacted in the crypts and HEK293T cells, respectively. To decipher the role of CREPT on β-catenin, we examined the protein level of β-catenin. A Western blot showed that the level of β-catenin in the nucleus was substantially decreased in CREPT KO cells (Fig. 6g). These results suggest that CREPT might regulate the retaining of β-catenin in the nucleus.

a–d CREPT deletion impaired the SuperTop luciferase activity stimulated by Wnt1 (a), APC depletion (b), β-catenin (c), and CHIR99021 (d). CREPT was deleted by a CRISPR-Cas9 system in HEK293T cells. SuperTop-luciferase reporter and pRL-TK plasmids transfected WT and CREPT deleted HEK293T cells were co-transfected with Wnt1 (a), shAPC (b), and β-catenin (c) plasmids, or treated with 5 μM CHIR99021 for 4 h (d). The activity was expressed as fold-changes, normalized by an internal control (Renilla). p < 0.0001 (a). p = 0.0168 (b). p = 0.0009 (c). p = 0.0002 (d). n = 3 independent experiments for each treatment. e Endogenous CREPT interacts with β-catenin. Cell lysis of intestinal crypts was incubated with control IgG or anti-CREPT antibodies. The immunoprecipitants were analyzed by Western Blotting using anti-β-catenin or anti-CREPT antibodies. f Exogenous CREPT interacted with β-catenin. Myc-tagged CREPT (Myc-CREPT) and FLAG-tagged β-catenin (Flag-β-catenin) were co-expressed in HEK293T cells. g CREPT deletion reduced the nuclear protein level of β-catenin. CREPT was deleted by the CRISPR-Cas9 system in DLD1 colorectal tumor cells. β-catenin proteins were analyzed by Western blotting in cytoplasmic and nuclear fractions of WT and CREPT KO DLD1 cells. h–k Representative images of β-catenin IHC staining in crypts at 0, 1, 3, and 5 dpi after X-ray irradiation, respectively. Obvious nuclear β-catenin appeared at 3 dpi of WT mice, but not Vil-CREPT^KO mice. l Quantification of nuclear β-catenin positive crypts per 1 mm intestine at 3 dpi. p < 0.0001. n = 3 mice per genotype. Images are representative of at least three independent experiments (n = 3 mice per genotype). Statistics data represent mean ± SEM. All p values were generated by 2-tailed Student’s t-test. Scale bars: 20 μm (h–k).

To confirm the role of CREPT on retaining β-catenin in the nucleus in vivo, we performed immunohistochemistry experiments. β-catenin was barely observed in the nucleus at 0 and 1 dpi of irradiated WT and Vil-CREPT^KO mice (Fig. 6h, i). Interestingly, overt nuclear β-catenin was detected in the intestinal crypts of irradiated WT mice at 3 dpi (Fig. 6j), and disappeared at 5 dpi (Fig. 6k), suggesting that Wnt signaling was activated at 3 dpi but terminated at 5 dpi during crypt regeneration. However, few cells had β-catenin in the nucleus in the crypts of irradiated Vil-CREPT^KO mice in the whole regeneration processes from 1 to 5 dpi (Fig. 6h–k). A quantitative analysis showed the amount of nuclear β-catenin was significantly reduced when CREPT was deleted at 3 dpi (Fig. 6l). All these results suggest that CREPT regulates the nuclear retaining of β-catenin in crypt cells during intestinal regeneration.