Search
Close this search box.

Claudin-7 is essential for the maintenance of colonic stem cell homoeostasis via the modulation of Wnt/Notch signalling – Cell Death & Disease

Cldn-7 is essential for maintaining the homoeostasis of Lgr5+ ISCs

We previously generated IEC-specific Cldn-7 knockout mice (Cldn-7fl/fl;Villin-CreERT2 mice) [27]. The Villin promoter targets Cre expression throughout the intestinal epithelium. Upon Tamoxifen (TAM) administration, Cldn-7 knockout mice exhibited rapid weight loss and intestinal swelling, particularly in the small intestine (Fig. 1A, B). Histopathological staining revealed severe damage to the colon, high infiltration of inflammatory cells, and disruption to the crypt architecture. While most crypts exhibited reduced proliferation, a compensatory increase was observed in a few crypts (Fig. 1B). Given that ISCs are self-renewing cells, crypt destruction in Cldn-7 knockout mice suggests the specific interference of Cldn-7 in the maintenance and function of crypt stem cells.

Fig. 1: Cldn-7 deletion in intestinal epithelial cells results in the loss of Lgr5+ stem cells and impairs differentiation capacity.
figure 1

A Body weight curve of mice during TAM induction for 5 days of Cldn-7fl/fl;Villin-CreERT2 and Cldn-7fl/fl;Villin-CreW mice (n = 5 per genotype). B Representative images of macroscopic intestine analysis, HE staining, and staining of colonic sections with the proliferation marker Ki-67; scale bars: 50 μm. C Representative images of Lgr5 mRNA (red) and Cldn-7 mRNA (green) detected by in situ hybridisation of distal colonic sections from Cldn-7-deficient mice and littermate controls (Left); the white arrow denotes Lgr5-positive cells at the bottom of the colonic crypt. Corresponding statistical analysis of Lgr5-positive cells in the crypt base is shown in the right panel (scale bars: 20 μm; n = 3 per genotype). D, E Quantitative RT-PCR analysis of marker genes for ISCs (Lgr5, Ascl2, Hopx, Lrig1, and Sox9) and differentiated cells (Anpep, Villin, Muc2, ChgA, and Dclk1) in colonic tissues from Cldn-7fl/fl;Villin-CreERT2 and Cldn-7fl/fl;Villin-CreW mice 5 days after induction (n = 6–8 per genotype). F Immunohistological staining of colonic sections to detect enterocytes (Villin), enteroendocrine cells (ChgA), goblet cells (Muc2, Alcian blue), and tuft cells (Dclk1); scale bars: 20 μm. For each genotype, the percentage or number of positive cells per field was calculated from 15 fields in 3 mice (Bottom panel). All images are representative of at least three independent experiments. Statistical data are presented as mean ± SEM. All p values were calculated using Student’s t-test (two-tailed) or Mann-Whitney nonparametric test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns, not significant.

To further explore this hypothesis, we assessed the expression levels of the ISC-specific marker, Lgr5, in the colonic epithelium. RNAscope results indicated that Lgr5 was localised to the crypt base of the colonic mucosa. IEC-specific Cldn-7 deficiency resulted in the downregulation of Lgr5 expression, with the loss of Lgr5 being more pronounced after DSS administration, highlighting the importance of Cldn-7 in ISC maintenance (Fig. 1C). Additionally, analysis of mRNA levels for various stem cell markers (Lgr5, Ascl2, Hopx, Lrig1, and Sox9) [3, 6, 8, 28, 29] revealed significant downregulation following Cldn-7 knockout (Fig. 1D), supporting the understanding that loss of Cldn-7 induces damage to Lgr5+ ISCs.

With Cldn-7 depletion reducing the ISC population, there is an insufficient activation of stem cells to compensate for continued differentiation. Histological staining and quantitative RT-PCR (qRT-PCR) analysis confirmed that Cldn-7 deficiency impaired the differentiation potential of ISCs, affecting the terminal differentiation of enterocytes, goblet cells, enteroendocrine cells, and tuft cells (Fig. 1E, F).

Cldn-7 null mice exhibit colonic stem cell dysfunction

To understand the cellular and molecular effects of Cldn-7 deletion on colonic epithelial morphogenesis, we performed RNA-seq analysis on colon tissue extracted from Cldn-7fl/fl;Villin-CreERT2 and Cldn-7fl/fl;Villin-CreW mice. The volcano plot illustrates the 929 upregulated genes and 1378 down-regulated genes identified in the Cldn-7 deletion group (Supplementary Fig. 1A-C). Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed associations with inflammatory activation, confirming the identified link to severe intestinal inflammation, alongside down-regulation of protein digestion and absorption (Supplementary Fig. 1D). Gene set enrichment analysis (GSEA) identified significant negative enrichment in Lgr5+ stem cell-, goblet cell-, enterocyte-, and tuft cell-related genes in Cldn-7 knockout tissues, accompanied by positive enrichment of inflammatory responses and apoptosis (Fig. 2A-D). ISC-related genes, such as Lgr5, Ascl2, Lrig1, Sox9, and Tert, were downregulated, whereas quiescent reserve ISC markers Bmi1 and Mex3a were upregulated. Differentiated cell markers, including Alpi, Anpep, Muc2, Dclk1, Kit, and Reg4, were also significantly downregulated (Fig. 2E, F), aligning with our PCR results.

Fig. 2: RNA sequencing and organoid culture analysis reveal colonic stem cell dysfunction in Cldn-7 null mice.
figure 2

AD Normalised enrichment scores (NES) from GSEA and enrichment plots for intestinal stem cells, differentiated cells, inflammatory responses, and apoptosis transcriptional signatures of Cldn-7fl/fl;Villin-CreERT2 and Cldn-7fl/fl;Villin-CreW colonic tissues, respectively. E Heatmap showing the expression levels of established genes in Wnt signalling pathways, stem cells, and mature differentiated cells in either Cldn-7fl/fl;Villin-CreERT2 or Cldn-7fl/fl;Villin-CreW colonic tissues. F Differential expression analysis of Log2 fold change in stem cells and Wnt signalling pathway by Cldn-7 deficiency. G Representative images of colonic organoids derived from Cldn-7fl/fl;Villin-CreERT2 and Cldn-7fl/fl;Villin-CreW mice (Left) from a minimum of three independent experiments; scale bars: 50 μm. The right panel shows the quantification of the average colonic organoid size on day 7 of the first passage. Organoids size was randomly measured in 50 organoids. H Quantitative RT-PCR analysis of marker genes for ISCs and differentiated cells in organoids derived from control mice and Cldn-7 knockout mice. (n = 3–4 per genotype). All images are representative of at least three independent experiments. Statistical data are presented as mean ± SEM. All p values were calculated using Student’s t-test (two-tailed) or Mann–Whitney nonparametric test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns, not significant.

Next, we analysed pathways closely linked to ISC niche maintenance, proliferation, and differentiation, including the Wnt pathway. Although the Wnt signalling pathway was not significantly enriched, Cldn-7 deletion downregulated the expression of genes downstream of the Wnt signalling pathway, including Axin2, Agr3, Ascl2, Jun, Wnt2b, and Wnt5b. The canonical Wnt target gene and stem cell marker Lgr5 was also significantly downregulated in the intestinal tissue of Cldn-7fl/fl;Villin-CreERT2 mice (Fig. 2E, F).

Subsequently, we employed colonic organoid cultures [30] to further investigate the potential of isolated colonic crypts to form clonal multifunctional organoids in the absence of Cldn-7. Compared with Cldn-7fl/fl;Villin-CreW mice, Cldn-7fl/fl;Villin-CreERT2 mice exhibited significantly reduced organoid formation efficiency, organoid diameter, and secondary organoid formation ability (Fig. 2G). qRT-PCR results demonstrated that ISC and differentiation marker genes were significantly downregulated (Fig. 2H), indicating defective organoid formation and differentiation capacities in Cldn-7-deficient crypts.

Cldn-7 deficiency in ISCs limits Lgr5+ ISC expansion during colonic homoeostasis

To further assess the impact of Cldn-7 deletion on the Lgr5+ ISC population, we established ISC conditional Cldn-7 knockout mice (Cldn-7fl/fl;Lgr5-CreERT2) and control mice (Cldn-7fl/fl;Lgr5-CreW). EGFP expression is driven by the endogenous Lgr5 promoter, allowing visualisation of Lgr5+ ISCs at the crypt base by detecting EGFP expression [3]. Immunofluorescence results revealed co-expression of EGFP and Cldn-7 in the colonic crypt (Fig. 3A). TAM was administered for 5 days to induce Cldn-7 deletion. RNAscope and immunofluorescence confirmed the significant loss of Cldn-7 expression in the crypt region. Moreover, the number of Lgr5-EGFP+ ISCs was significantly reduced after Cldn-7 deletion (Fig. 3B, C). Subsequently, colonic crypts were isolated from TAM-or vehicle control (VEH)-treated Cldn-7fl/fl;Lgr5-CreERT2 mice for single-cell flow cytometry, results revealed downregulation of Lgr5-EGFP+ ISCs in the TAM treatment group (Supplementary Fig. 2). Cldn-7 expression in sorted EGFP+ cells treated with TAM decreased by approximately 85%, confirming the efficient deletion of the Cldn-7 allele in ISCs (Fig. 3D). Moreover, during TAM induction, Cldn-7fl/fl;Lgr5-CreERT2 mice showed aggravated weight loss and shortened colon length (Fig. 3E).

Fig. 3: Cldn-7 deficiency reduces Lgr5+ ISC numbers and proliferative capacity in vivo.
figure 3

A Representative fluorescence images of Lgr5-EGFP and Cldn-7 staining of intestinal crypts. Arrowheads denote Cldn-7 expression (red) in Lgr5+ ISCs (EGFP); scale bars: 20 μm. B RNAscope analysis confirms the effects of Cldn-7 deletion (green) in the crypt region, detected across a minimum of three independent replicates; scale bars: 20 μm. C Immunofluorescence images of distal colonic tissues stained for Cldn-7 (red) and Lgr5-EGFP (green); cell nuclei were counterstained with DAPI (blue). The arrow indicates the absence of Cldn-7 in the crypt area; scale bars: 20 μm. Graphs (right) present the quantification of Lgr5+ ISCs in the intestinal crypts (n = 3 per genotype). D Quantitative RT-PCR analysis of Cldn-7 in sorted Lgr5-EGFP+ ISCs by flow cytometry in colonic tissues from Cldn-7fl/fl;Lgr5-CreERT2 mice after VEH or TAM administration. (n = 5 per genotype). E Changes in body weight (n = 7 per genotype) and colon length (n = 9 per genotype) during TAM induction. F Representative images of pathological HE staining, Ki-67 staining, and TUNEL assays of colon sections from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice following TAM induction for 5 days (D5) and recovery for 1 week (D12). Arrowheads denote intestinal epithelial disruption. Images are representative of 3 mice per genotype; scale bars: 50 μm. Right panels: quantification of Ki-67-positive cells per crypt base and TUNEL-positive cells per field. G RNAscope in situ hybridisation of Lgr5 (red) and Cldn-7 (green) in the colonic mucosa from the two groups of mice during injury and recovery periods; scale bars: 20 μm. The right panels show the quantification of the Lgr5 signal in each field of tissue at different time periods (n = 3–4 per genotype). H Quantitative RT-PCR analysis of the indicated stem cell markers in colon tissues from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice at D5 and D12 (n = 4–5 per genotype). Statistical data are presented as mean ± SEM. All p values were calculated using Student’s t-test (two-tailed) or Mann–Whitney nonparametric test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns, not significant.

Upon induction of Cldn-7 deficiency, the intestinal epithelial mucosal layer was disrupted, with a slight increase in the number of inflammatory cells present. Next, we performed several assays to determine whether the destruction of colonic epithelial cells and loss of Lgr5+ ISCs could be attributed to reduced cell survival. Overall, the number of Ki67+ proliferating cells was lower under Cldn-7 deficiency. In Cldn-7fl/fl;Lgr5-CreW colonic mucosa, minimal apoptosis of terminally differentiated epithelial cells was observed on the luminal surface; conversely, Cldn-7fl/fl;Lgr5-CreERT2 mice displayed substantial apoptosis in both the terminal epithelium and the crypt base columnar region. With the continuous renewal of the intestinal epithelium, the proliferation and apoptosis of Cldn-7fl/fl;Lgr5-CreERT2 mice were up-regulated compared with those after TAM induction (Fig. 3F). Meanwhile, in situ hybridisation results revealed strong Lgr5 expression in colonic crypt base cells in the control group, which was significantly reduced after Cldn-7 deletion. Even after one week of recovery, Lgr5 expression remained downregulated compared to that in the Cldn-7fl/fl;Lgr5-CreW group (Fig. 3G). qRT-PCR results confirmed decreased expression of ISCs markers following Cldn-7 deletion. However, mRNA levels of Lgr5, Lrig1, and Sox9 in the colon crypts started to recover at D12 post-TAM induction, whereas Ascl2 and Hopx expression gradually increased, suggesting that the emergence of other reserve stem cell populations may compensate for the initial loss of Cldn7-null ISCs (Fig. 3H).

Cldn-7 loss in Lgr5+ ISCs disrupts differentiation of colonic stem cells

To assess the impact of Cldn-7 depletion on intestinal cell differentiation, we analysed the abundance of mature differentiated cells in the colonic epithelium. Among the intestinal epithelial cells, enterocytes were the most abundant; however, immunofluorescence staining showed that the expression of the enterocyte marker gene was significantly downregulated (Fig. 4A). Localisation analysis of goblet cells using alcian blue staining and immunohistochemical staining for Muc2 revealed a significant reduction after TAM treatment (Fig. 4B, C). Additionally, the expression of enteroendocrine cells and tuft cells was also downregulated (Fig. 4D, E).

Fig. 4: ISC-specific Cldn-7 loss disrupts epithelial differentiation in vivo.
figure 4

Immunohistochemical and immunofluorescence staining were used to detect the abundance of colonic epithelial differentiated cells in Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice following TAM induction (D5) and 1 week of recovery (D12). A Villin marks enterocytes. B, C Alcian blue and Muc2 mark goblet cells. D ChgA marks enteroendocrine cells. E Dclk1 marks tuft cells. Nuclei were counter-stained with haematoxylin or DAPI. Images are representative of n = 3–4 mice per genotype; scale bars: 50 μm. Quantifications of the number or percentage of positive cells for each differentiated cell are shown beside each representative image. F, G Quantitative RT-PCR analysis of the indicated differentiated cell markers in colonic tissues from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice at D5 and D12 (n = 4–5 per genotype). Statistical data are presented as mean ± SEM. All p values were calculated using Student’s t-test (two-tailed) or Mann–Whitney nonparametric test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns, not significant.

Notably, we discovered an interesting phenomenon, an increase in the abundance of secretory lineage cells, such as goblet and enteroendocrine cells, was observed during damage repair of the colonic epithelium in the Cldn-7 deletion group; however, the abundance of absorptive lineage enterocytes remained low in the regenerative colonic epithelium. The results of qRT-PCR showed that the Villin+ cells in the Cldn-7fl/fl;Lgr5-CreERT2 mice were down-regulated at both the injury and repair period, while the secretory lineage cells (Muc2+ goblet cells, ChgA+ enteroendocrine cells) were down-regulated at the injury period and significantly increased at the repair stage (Fig. 4F, G).

Cldn-7 is essential for growth and differentiation of colonic organoids

To further analyse the functional relevance of Cldn-7 and ISCs, we treated Cldn-7fl/fl;Lgr5-CreERT2 mice with TAM or VEH; then, colonic crypts derived from both groups were used to generate colonic organoids via in vitro culture. Quantitative analysis revealed that TAM-treated organoids exhibited slower growth, reduced diameter, and a significantly reduced organoid formation efficiency. The growth kinetics of the organoids after passage were also significantly different, with the Cldn-7 deletion group displaying significantly downregulated amplification efficiency and reduced formation of crypts (Fig. 5A, B).

Fig. 5: Cldn-7 is necessary for the growth and differentiation of colonic organoids in vitro.
figure 5

A Representative bright field images of colonic organoids generated from Cldn-7fl/fl;Lgr5-CreERT2 mice treated with VEH or TAM at primary (P0) and first passage (P1); scale bars: 100 μm. B Statistical plots of the organoid-forming efficiency of primary cultures, average organoid sizes at day 8 of primary culture, and the replating efficiency at day 5 after first passage. All experiments were repeated three times with at least three mice in each group. C Representative image from Lgr5-EGFP+ ISCs in VEH- and TAM-treated Cldn-7fl/fl;Lgr5-CreERT2 mice, visualised by fluorescent confocal microscopy 5 days after passage. Lgr5-EGFP immunofluorescence is shown in green (white arrowheads); scale bars: 100 μm. The right panel shows the quantification of Lgr5-EGFP+ ISCs per organoid. D, E Representative images of colonic organoids in each group; samples were stained with HE, alongside Ki-67 (green) to detect proliferation, TUNEL (red) to detect apoptosis, Villin (red) to detect enterocytes, Muc2 (red) and Alcian blue to detect goblet cells, ChgA (red) to detect enteroendocrine cells, and Dclk1 (red) to detect tuft cells. Blue represents DAPI staining of nuclei; scale bars: 50 μm. Images are representative of at least three mice. FH Statistical quantification of Ki-67, TUNEL, Villin, Muc2, ChgA, Dclk1-positive cells per organoid. Statistical data are presented as mean ± SEM. All p values were calculated using Student’s t-test (two-tailed) or Mann–Whitney nonparametric test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns, not significant.

Next, we assessed the number of Lgr5-EGFP+ ISCs using confocal microscopy. Organoids receiving TAM therapy had fewer crypt structures and exhibited a weak green fluorescence signal; therefore, Cldn-7 deletion can lead to a reduction in Lgr5-EGFP+ ISCs (Fig. 5C). Meanwhile, the mRNA expression levels of stem cell-associated genes decreased at the organoid level, further confirming that the stemness of Cldn-7-deficient colonic organoids was inhibited (Supplementary Fig. 3A).

Immunohistochemical examination and qRT-PCR analysis revealed that these organoids replicated the in vivo composite phenotype, with reduced proliferation and increased apoptosis of organoids following Cldn-7 deletion (Fig. 5D, F). The expression of intestinal differentiation markers for enterocytes (Villin), goblet cells (Muc2) and enteroendocrine cells (ChgA) were downregulated (Fig. 5D, E, G, H). qRT-PCR revealed reduced transcription of differentiated cell marker genes in Cldn-7 deficient colonic organoids (Supplementary Fig. 3B).

Cldn-7 mediates susceptibility to experimental colitis and contributes to intestinal epithelial regeneration

As Cldn-7 depletion resulted in a reduced self-renewal capacity of ISCs, we investigated the role of Cldn-7 in intestinal inflammation and damage repair. To induce colonic epithelial ulceration and inflammation, Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice were treated with 2%DSS for 7 days to induce acute colitis, followed by normal drinking water for 14 days to observe the repair of intestinal injury. After 4 days of treatment, DSS-exposed Cldn-7fl/fl;Lgr5-CreERT2 mice showed more pronounced weight loss and displayed clinical symptoms of hypoactivity and lethargy (Fig. 6A–C).

Fig. 6: Cldn-7 affects susceptibility to experimental colitis and contributes to intestinal epithelial regeneration.
figure 6

A Schematic diagram showing the injury and regeneration phases of experimental colitis in adult Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice after TAM induction for 5 days. B Changes in body size of mice in each group after 3% DSS exposure. C Weight loss curves of Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice during the 5 days of TAM induction and 7 days of DSS treatment (n = 5 per genotype). D Representative images of colon length changes in mice following DSS induction (Injury) and 2 weeks of recovery (Regeneration). The right panel indicates the corresponding statistical data for colon length analysis (n = 5 per genotype). E Representative histological HE (top), Ki-67 (middle), and TUNEL (bottom) staining images of colonic tissues from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice during acute inflammatory injury (7 days of DSS treatment) and regeneration (7 days of DSS followed by 14 days of regular water consumption). Arrowheads denote extensive mucosal defects and crypt destruction; scale bars: 50 μm. The right panels show the quantification of Ki-67-positive and TUNEL-positive cells in injured and regenerating epithelial tissue (three slides per sample; n = 3–4 per genotype). F, G Quantitative RT-PCR analysis of ISC-related marker genes in intact colonic tissues during intestinal inflammatory injury and regeneration, respectively, in each group of mice. All images are representative of at least three independent experiments. Statistical data are presented as mean ± SEM. All p values were calculated using Student’s t-test (two-tailed) or Mann–Whitney nonparametric test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns, not significant.

Macroscopic examination of the dissected colon after DSS administration revealed significantly shortened colon lengths and the presence of dark red bloody stools in the intestinal lumen, indicating more severe inflammation in Cldn-7 knockout mice than in control mice (Fig. 6D). Moreover, the colons of Cldn-7 deficient mice displayed damage to the colonic mucosa, complete absence of crypt structures, and infiltration of a large number of immune cells; proliferating cells were scarce in the intestinal epithelium, and apoptosis was significantly upregulated in all layers of the colonic epithelium. In contrast, the control group exhibited a small number of residual crypts and increased cell proliferation/apoptosis ratio (Fig. 6E). qRT-PCR results indicated a significant decrease in ISC markers in the Cldn-7 deletion group following DSS treatment, indicating that Cldn-7 deletion further impaired ISC maintenance (Fig. 6F).

To further understand the impact of Cldn-7 on intestinal epithelial regeneration, we collected colonic tissue 14 days after DSS injury. In both groups, the intestinal structure underwent continuous remodelling. With the expansion of crypts, the cells exhibited compensatory proliferation extending beyond the crypts into the lower 2/3 of the intestinal epithelium; additionally, apoptosis levels were significantly reduced. However, intestinal recovery was slower in the Cldn-7 knockout group than in the control group, with a lower proliferation level and continued down-regulation of ISC-related factors (Fig. 6E, G).

Increased secretory lineage differentiation in DSS-induced Cldn-7-deficient colonic epithelium

Next, we explored the effects of Cldn-7 depletion on epithelial differentiation during acute inflammation and wound repair. Consistent with the loss of epithelial differentiation following acute Cldn-7 loss, the differentiation ability of intestinal epithelial cells was also impaired under inflammatory conditions. Nonetheless, increased secretory lineage differentiation was observed in the Cldn-7 depletion group during enteritis induction. Specifically, the numbers of enterocytes and tuft cells significantly decreased (Fig. 7A, B), while the numbers of enteroendocrine and goblet cells increased (Fig. 7C, D). Moreover, the colonic epithelium of Cldn-7 knockout mice exhibited sustained high abundances of secretory cell lineages during tissue injury and repair. qRT-PCR results also confirmed these findings that the secretory cell lineage was upregulated and the absorptive lineage was downregulated (Fig. 7E, F).

Fig. 7: Increased secretory lineage differentiation by DSS-induced Cldn-7-deficient colonic epithelial cells.
figure 7

Immunofluorescence staining was used to detect the expression of colonic epithelial differentiated cells in Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice during acute injury (DSS d7) and regeneration (DSS d7 + H2O d14). AD Representative images of (A) Villin staining (enterocytes), (B) Dclk1 staining (tuft cells), (C) ChgA staining (enteroendocrine cells), and (D) Muc2 staining (goblet cells), with the lower panels showing the local magnified view of Muc2 staining within the boxes. Nuclei were counter-stained with DAPI; scale bars: 50 μm. Quantifications of the number or percentage of each differentiated cell type during the injury and regeneration period are shown in the right panels (n = 3-4 per genotype). E, F Quantitative RT-PCR analysis of the indicated differentiated cell markers in colon tissues from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice during injury and regeneration, respectively (n = 35 per genotype). All images are representative of at least three independent experiments. Statistical data are presented as mean ± SEM. All p values were calculated using Student’s t-test (two-tailed) or Mann–Whitney nonparametric test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns, not significant.

Cldn-7 promotes colonic epithelial homoeostasis and regeneration through Wnt and Notch signalling

To gain a comprehensive understanding of the underlying mechanisms by which Cldn-7 maintains colonic homoeostasis, we conducted sequencing analysis of the colonic epithelium from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice. RNA-seq results identified 702 differentially expressed genes (DEGs) (padj ≤ 0.05, log2 fold change ≥ 1.0), among which 291 were upregulated and 411 were downregulated; notably, the number of DEGs was significantly lower in IEC-specific Cldn-7 knockout mice (Supplementary Fig. 4A, B). The ISC signature genes and differentiated cell lineage genes were significantly downregulated in ISC-specific Cldn-7 knockout mice (Fig. 8A). To analyse the transcriptional changes associated with Cldn-7 loss in ISCs, we performed KEGG and GSEA to compare our RNA-seq dataset with previously established signature gene sets. Lgr5+ stem cell gene signalling [4], Wnt signalling [31], and Notch signalling [32] were significantly enriched in downregulated genomes (Fig. 8B, Supplementary Fig. 4C). This simultaneous inhibition of both Wnt and Notch signalling further supported the increased propensity for differentiation into secretory cell lineages [9, 33], consistent with our histological findings.

Fig. 8: Cldn-7 promotes colonic epithelial homoeostasis and regeneration through the Wnt and Notch signalling pathway.
figure 8

A Differential expression analysis showing the Log2 fold change values of stem cells and differentiated cells. B Representative GSEA enrichment plots showing the negatively enriched gene sets for Lgr5+ stem cells, Wnt signalling, and Notch signalling in Cldn-7fl/fl;Lgr5-CreERT2 mice. C Quantitative RT-PCR verification of Wnt signalling related genes (Axin2, β-catenin, C-myc, Wnt3, Wnt5b, Cyclind1, Ephb2, Fzd5, Fzd7, Tcf4, and CD44) and Notch signalling related genes (Dll1, Dll4, Hes1, and Notch1) in colonic tissues from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice treated with TAM for 5 days. n = 4–6 per genotype. D Immunostains and quantitative analysis of β-catenin, Notch1 and Hes1 labelling in colonic tissues from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice treated with TAM for 5 days, scale bars: 50 μm, n = 3 per genotype. E Immunoblot analysis showing the expression of specific proteins (Cldn-7, β-catenin, C-myc, and Cyclind1) in the colon from Cldn-7fl/fl;Lgr5-CreERT2 and Cldn-7fl/fl;Lgr5-CreW mice treated with TAM for 5 days. β-actin was employed as a loading control; n = 3 per genotype. Data are representative of three independent experiments. F Quantitative RT-PCR verification of Wnt and Notch signalling-related genes in colonic organoids derived from Cldn-7fl/fl;Lgr5-CreERT2 mice that were treated with VEH or TAM (n = 3 per genotype). G Colonic organoids derived from Cldn-7fl/fl;Lgr5-CreERT2 crypts (VEH or TAM treatment) that were treated with DMSO, Chir (10 μM), Wnt3a (100 ng/mL), or Wnt3a+Chir for 48 h. Quantitative RT-PCR analysis was used to detect β-catenin, C-myc, Cyclind1, and Wnt3 expression in these organoids (n = 3 per genotype). H Representative images of colonic organoids derived from Cldn-7fl/fl;Lgr5-CreERT2 crypts (VEH or TAM treatment) were treated with DMSO, Chir (10 μM), Wnt3a (100 ng/mL), or Wnt3a+Chir; scale bars: 50 μm. Right panel: Quantification of average organoid size per organoid for Cldn-7fl/fl;Lgr5-CreERT2 with different treatments. Statistical data are presented as mean ± SEM. All p values were generated by Student’s t-test (two-tailed) or Mann–Whitney nonparametric test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns, not significant.

We further examined the mRNA expression levels of Wnt and Notch signalling pathway-related factors, most of which were downregulated following Cldn-7 deletion (Fig. 8C). Additionally, protein expression levels of key Wnt signalling proteins, including β-catenin, C-myc, and Cyclind1, as well as the Notch receptor Notch1 and Notch target Hes1 were significantly reduced (Fig. 8D, E). Similar results were observed in colitis-induced tissues, where transcription levels and protein levels of Wnt and Notch signalling pathway-related factors were significantly reduced after Cldn-7 depletion (Supplementary Fig. 5A–C). This confirmed that Cldn-7 deletion inhibited transcriptional regulation of the Wnt/Notch signalling pathway, leading to a reduction in stem cells and an increased propensity for differentiation into secretory cells.

To further determine the role of Cldn-7 in signalling regulation, we cultured colonic crypts treated with TAM or VEH to produce colonic organoids. Cldn-7 deficiency was found to inhibit Wnt and Notch signalling at the organoid level, with significant down-regulation of Wnt pathway target genes and Notch pathway-related factors (Fig. 8F). Treatment of cultured organoids with exogenous Wnt3a or GSK3β inhibitor CHIR99021 (Chir), known to promote ISC function [34, 35], revealed that Wnt pathway-related genes were up-regulated in Cldn-7-deficient organoids after Wnt-activating factor treatment (Fig. 8G). The combined Wnt3a and Chir treatment significantly increased the branching of VEH-treated colonic organoids and promoted growth. However, combined treatment with Wnt3a and Chir did not promote TAM-treated colonic organoids to the same extent (Fig. 8H). Overall, this confirms that stimulation of β-catenin via exogenous addition of Wnt3a or Chir can partially rescue the growth defect phenotype of Cldn-7 knockout organoids; nonetheless, Cldn-7 is essential for ISC maintenance and Wnt signalling activation.

Latest Intelligence