Skip to main content
Download PDF
  • Research Article
  • Open access
  • Published:

WNT4 promotes the symmetric fission of crypt in radiation-induced intestinal epithelial regeneration

Cellular & Molecular Biology Letters volume 29, Article number: 158 (2024) Cite this article

Abstract

Background

Radiotherapy for pelvic malignant tumors inevitably causes intestinal tissue damage. The regeneration of intestinal epithelium after radiation injury relies mainly on crypt fission. However, little is known about the regulatory mechanisms of crypt fission events.

Methods

The effects of WNT4 on crypt regeneration and the symmetry of crypt fission were examined using a mouse small intestinal organoid culture model. Three-dimensional (3D) reconstructed images of organoids were applied to assess the symmetry of crypt fission and Paneth cell localization upon manipulation of WNT4 expression. The effect of WNT4 on the expression of β-catenin target genes was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). The in vivo effect of WNT4 overexpression mediated by adeno-associated virus (AAV) on symmetric fission of crypt was investigated using a radiation-injured mouse model.

Results

WNT4 has a special function of promoting symmetric fission of small intestinal crypts, although it inhibits budding, stemness, and cell proliferation on organoids. WNT4 promotes the correct localization of Paneth cells in the crypt base by regulating the expression of EphB3, thereby promoting the symmetric fission of small intestinal crypts. WNT4 negatively regulates the canonical WNT/β-catenin signaling pathway, and it promotes symmetric crypt fission in a ROR2 receptor-dependent manner. Moreover, in patients and animal models of radiation-induced intestinal injury, we found that the regenerated crypts are irregular in size and shape, Paneth cells are mislocalized, and the expression of WNT4 is decreased while EphB3 is increased. Importantly, restoration of WNT4 expression mediated by AAV effectively promotes symmetric crypt fission and thus improves the regularity of regenerating crypts in mice with radiation-induced injury.

Conclusions

Our study highlights the critical role of WNT4 in the regulation of crypt fission and provides WNT4 as a potential therapeutic target for radiation enteritis.

Graphical Abstract

Introduction

The intestinal epithelium is characterized by rapid self-renewal, which occurs every 4–5 days [1, 2]. Intestinal stem cells (ISCs) located at the crypt base give rise to transit-amplifying progenitor cell population [3]. These precursors further differentiate into different cell lineages and follow a bidirectional migration path, that is, enterocytes, goblet cells, and enteroendocrine cells migrate up to the villus, while Paneth cells migrate downward and reside at the bottom of the crypt [4]. Eph/Ephrin signaling plays a central role in directing cell migration, and disruption of EphB3 signaling results in misposition of Paneth cells, which do not migrate downward but scatter along the crypt-villus axis [5, 6].

Crypt fission is an important process of intestinal tissue expansion during postnatal development [7]. The number of crypts increases in the form of fission, in which a single crypt divides into two daughter crypts [8]. The ways of crypt fission include symmetry and asymmetry [9,10,11]. The symmetric fission is characterized by a bifurcation appearing in the middle of the crypt base, which extends upward to form two daughter crypts of equal length, while the initial bifurcation of asymmetric fission is asymmetrically placed at the crypt base or above the crypt base, and eventually forms two branches of unequal length [12]. Intestinal organoids maintain the structural characteristics of normal intestinal epithelium, and the formation of new branches during the growth of organoids mimics the crypt fission in intestinal tissue [12, 13]. There are two cell types in the crypt base, Lgr5+ stem cells and Paneth cells, which play distinct roles in the fission process [12, 14]. It has been reported that the location of Paneth cells determines the initiation site of crypt fission, and the mislocation of Paneth cells drives asymmetric fission [12, 15].

The WNT family plays critical roles in intestinal epithelium [16,17,18,19]. The canonical WNT/β-catenin cascade is essential for homeostasis and regeneration of intestinal epithelium [20]. WNT/β-catenin signaling also regulates the expression of EphB3 to control the localization of Paneth cells [5]. The non-canonical WNT ligands signal via planar cell polarity (PCP) or calcium intracellular pathways, and can either activate or inhibit β-catenin pathway [21]. WNT5a has been reported to promote crypt fission in a TGF-β-dependent manner during regeneration after epithelial injury [22]. Another most widely studied typical WNT ligand, WNT4, is known to play key roles in the development and homeostasis of various organs [23]. However, whether WNT4 plays a role in intestinal epithelial regeneration, especially in the crypt fission process, is still unknown.

In clinical practice, intestinal tissue damage is inevitable when radiotherapy is performed on patients with pelvic malignant tumors [24], and the regeneration of intestinal epithelium after radiation injury largely depends on crypt fission [25]. However, how this regeneration process is regulated after radiation injury is poorly understood. Here, we applied the organoid model to reveal a particular role of WNT4 in promoting symmetric fission of crypts, and further evaluated the potential therapeutic value of adeno-associated virus (AAV)-delivered Wnt4 overexpressing in promoting intestinal tissue repair in a radiation injury mouse model.

Materials and methods

Patients and samples

In total, 18 specimens were obtained from patients (female, age range from 31 to 77 years) with radiation enteritis (RE) who had received radiation therapy with a total dose of 50–98 Gy for cervical cancer, were diagnosed with RE on the basis of pelvic radiotherapy history radiological and colonoscopic features as described previously [26], and underwent surgical resection of the affected segment of intestine. Then, seven paired RE specimens were obtained from resected RE lesions and paired adjacent non-injured intestinal tissues. Specimens were snap-frozen or formalin-fixed for further use immediately after acquisition.

Animal study

All animals were raised in specific pathogen-free conditions and maintained in a controlled temperature environment with a 12 h light/dark cycle. The radiation-induced intestinal injury mouse model was established using female C57BL/6J mice aged 8–10 weeks purchased from GemPharmatech Co., Ltd. (Nanjing, China). After anesthesia, mice received abdominal irradiation with a single dose of 10.5 Gy containing the gastrointestinal tract using a RS2000 X-ray Irradiator (Rad Source Technologies, Inc., Boca Raton, FL, USA), and the rest of the body including the thorax, head and neck, and extremities were shielded. Mice in the normal control group only received sham irradiation after anesthesia. Mice were euthanized 4 weeks after irradiation and small intestinal tissues were removed for histological examination.

For AAV treatment experiment, the mice were intraperitoneally injected with AAV-Wnt4 (AAV-CMV-Wnt4-FLAG-GdGreen, OBiO Technology Co., Ltd, Shanghai, China) or AAV-vector (AAV-CMV-GdGreen, OBiO Technology Co., Ltd, Shanghai, China) at a dose of 2 × 1011 vg/mouse on the next day after 10.5 Gy of abdominal irradiation. At 4 weeks after irradiation, the mice were sacrificed and the small intestine tissues were collected for further examination.

We defined symmetric and asymmetric crypt fission in mouse small intestinal tissue with reference to previous report [27]. Symmetric crypt fission: bifurcated crypt showing two daughter crypts of the same length, diameter, and shape connected by a single crypt lumen. Asymmetric crypt fission: branched crypt showing two or more daughter crypts with varying diameters, lengths, and shapes connected by a single crypt lumen.

Mouse crypt isolation and organoid culture

Mouse crypt isolation and organoid culture were performed as described previously [28]. Briefly, mouse small intestine was removed and opened longitudinally. After gently scraping the surface feces and mucus, the intestinal tissue was cut into 2-mm pieces and washed with cold phosphate buffered saline (PBS). Tissues were incubated in 5 mM ethylenediaminetetraacetic acid (EDTA) at 4 °C for 30 min. After incubation, the supernatant was removed, and the tissue pieces were resuspended in ice-cold D-PBS and vigorously triturated using a 25-ml pipette. The separated crypts in the supernatant were filtered through a 70-μm filter into a new tube [precoated with 1% bovine serum albumin (BSA)] and then centrifuged at 500 g for 5 min. The pellet crypts were then resuspended in Matrigel (BD Biosciences, San Jose, CA, USA) and transferred to 24-well cell culture plate. Mouse IntestiCult™ Organoid Growth Medium (Stemcell Technologies) was added after the Matrigel was polymerized and changed every 2 days.

Lentivirus transfection

For the preparation of lentiviral particles,Wnt4-overexpressing plasmid (pEZ-Lv242-Wnt4, GeneCopoeia, Maryland, USA) or the control plasmid (pEZ-Lv242-vector, GeneCopoeia, Maryland, USA) were transfected in HEK 293FT cells together with lentiviral packaging vectors using PEI transfection solution. Supernatants containing virus were collected 48 and 72 h after transfection and centrifuged at 50,000 g for 90 min. The viral pellets were then resuspended in 500 μl of organoid culture medium supplemented with 10 μg/ml polybrene.

For lentiviral transduction of organoids, organoids were harvested by mechanically disrupting the Matrigel, and then digested with trypsin for 3 min at 37 °C. After removal of trypsin by centrifugation, the organoids were mixed with the virus solution and centrifuged on 32 °C at 600 g for 1 h in a 48-well plate to enhance transduction efficacy. The organoid-virus mixture was incubated in a culture incubator for 1 h at 37 °C, and then resuspended with 500 μl of organoid culture medium and centrifuged for 5 min at 500 g to pellet organoids. The organoid pellet was then embedded in 50 μl of ice-cold matrigel and cultured in 0.5 ml of mouse IntestiCult™ Organoid Growth Medium.

siRNA transfection

siRNA transfection of organoids was performed according to previous study [29]. The siRNA transfection solution was prepared by mixing Wnt4 siRNA (RiboBio Co. Ltd, Guangzhou, China) or control siRNA (RiboBio Co. Ltd, Guangzhou, China) with RNAiMAX (Invitrogen) in DMEM (Gibco) containing 10% FBS (Gibco), and was added directly to organoids after 2 days of culture in a 24-well plate. The medium was replaced with fresh organoid culture medium 48 h after transfection.

Histology, immunohistochemistry (IHC), and immunofluorescence (IF)

Human and mouse intestinal tissues were fixed with 4% paraformaldehyde and paraffin embed. Sections were stained with hematoxylin and eosin (HE).

IHC staining was carried out using SP-9000 SPlink Detection Kit (ZSGB-Bio, Beijing, PR China) following the manufacturer’s protocol. The following primary antibodies were used: anti-WNT4 (1:100, 14371-1-AP, Proteintech group), anti-Ki67 (1:300, ab16667, Abcam) and anti-lysozyme (1:300, ab108508-40, Abcam) antibodies.

For IF staining of tissue sections, permeabilization was performed with PBS containing 0.1% Triton X-100. After blocking with 1% BSA, the sections were incubated with primary antibodies at 4 °C overnight, washed and subsequently incubated with fluorescence-conjugated secondary antibodies Alexa Fluor 488 (1:1000, A-32723, Invitrogen) or Alexa Fluor 594 (1:1000, A-11037, Invitrogen) at room temperature for 1 h. Nuclei were counterstained with DAPI (Beyotime, Beijing, China). Confocal laser scanning was performed using a Leica TCS-SP8 confocal microscope (Mannheim, Baden-Wuerttemberg, Germany). The primary antibodies utilized in this study were anti-EphB3 (1:300, AF432-SP, R&D systems), anti-lysozyme (1:300, ab108508-40, Abcam), anti-WNT4 (1:100, 14371-1-AP, Proteintech group), anti-Flag (1:200, F1804, Sigma), and anti-ROR2 (1:8, Ror2, DSHB) antibodies.

For IF staining of organoids, organoids isolated from Matrigel were fixed with 4% paraformaldehyde at 37 °C for 45 min, permeabilized using 1% Triton-X100 for 1 h, and then incubated with blocking buffer (3% normal goat serum, 1% BSA and 0.2% Triton-X100 in PBS) for 1 h at room temperature. The following primary antibodies were used to incubate overnight with the organoids in Working Buffer (0.3% normal goat serum, 0.1% BSA, 0.2% Triton-X100 in PBS): anti-lysozyme (1:200, ab108508-40, Abcam), anti-Ki67 (1:200, ab16667, Abcam), anti-WNT4 (1:50, 14371-1-AP, Proteintech group), and anti-ROR2 (1:4, Ror2, DSHB) antibodies. After washing, AlexaFluor-conjugated secondary antibodies (1:1000, Invitrogen) along with Phalloidin (1:100, PF00003, Proteintech group) were applied overnight in Working Buffer. Nuclei were counterstained with DAPI (1:5000, Beyotime, Beijing, China) or Hoechst 33342 (1:5000, Beyotime, Beijing, China). After mounting with ProLong Gold antifade (Meilunbio), images were taken with Lecia TCS-SP8 or STELLARIS 8 FALCON confocal microscopes (Mannheim, Baden-Wuerttemberg, Germany).

Time series of live imaging of organoid culture

Live imaging of organoids was captured using Operetta CLS HCS (PerkinElmer, USA). Imaging processing was conducted using the Harmony 4.8 software (PerkinElmer, USA).

3D image processing and analysis

Organoids were imaged with a Lecia STELLARIS 8 FALCON confocal microscope (Mannheim, Baden-Wuerttemberg, Germany) using 40× objective lens, and Z stacks were taken at 1 μm steps. Measurements of crypt length and Paneth cell distance were based on 3D projections of organoids using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Symmetric fission in organoids was assessed according to previous report [12]. That is, a ratio of the length of the shorter to the length of the longer daughter crypt greater than 0.75 was considered a symmetric fission. The localization of Paneth cells was determined on the basis of staining for lysozyme, and the distance of each Paneth cell from the crypt base was measured to analyze their distribution.

RT-qPCR

Total RNA in tissues or organoids was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was carried out using ReverTra Ace Master Mix (TOYOBO, Osaka, Japan). Real-time quantitative polymerase chain reaction (RT-qPCR) was conducted with FastStart Essential DNA Green Master (Roche, Basel, Switzerland) using RT-PCR systems (Applied LightCycler96 Roche). GAPDH or ACTB served as the internal control. The primer sequences applied in this study are listed in Additional file 1, Supplementary Table 1.

Immunoprecipitation (IP)

IP assay was performed according to our previous study [28]. Briefly, whole small intestinal tissues from normal C57BL/6J mice (8–10 weeks, female) were lysed using lysis buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, 1% NP-40). Lysates were precleaned with 25 μl of protein G affinity gel (E3403, Sigma, St Louis, MO, USA) at 4 °C for 1 h. The supernatant was immunoprecipitated with 1 μg of anti-ROR2 antibody (Ror2, DSHB) or mouse immunoglobulin G (IgG) isotype control (MAB002, R&D systems) at 4 °C overnight, and then incubated with 30 μl of protein G gel at 4 °C for 1 h. The immunoprecipitated complex was washed with wash buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, 0.1% NP-40) six times, and eluted with 2× sample buffer, and then subjected to western blot analysis.

In situ hybridization (ISH)

The probe for Wnt4 was designed and synthesized by Servicebio (Wuhan, China). The probe sequences were as follows: 5′-TGAGTTTCTCGCACGTCTCCTCTT-3′, 5′-TCCACAAAGGACTGTGAGAAGGCTAC-3′, 5′-GGCTTTAGATGTCTTGTTGCACGTGC-3′.

Small intestines from mice at 4 weeks post-irradiation (10.5 Gy) and the normal control mice were fixed in formalin, embedded in paraffin and sliced into 4 μm sections. After being dewaxed and rehydrated, the sections were repaired in a citric acid antigen retrieval solution (pH 6.0) at 90 °C for 48 min and digested in proteinase K (20 μg/ml) at 40 °C for 10 min. After blocking endogenous peroxidase with 3% methanol –H2O2, sections were prehybridized at 37 °C for 1 h and hybridized in a solution containing the probes overnight at 40 °C. Sections were washed in 2 × SSC for 10 min at 37 °C, in 1 × SSC at 37 °C for 2 × 5 min and in 0.5 × SSC at room temperature for 10 min. Sections were then blocked in normal rabbit serum at room temperature for 30 min and incubated with anti-DIG-HRP at 37 °C for 50 min. After washing for several times with PBS, color development was performed with DAB and nuclei were counterstained with hematoxylin. Signal intensities in the crypt and villus regions were quantified separately with four random 200× magnification fields per slide using ImageJ software.

Statistical analysis

Data analyses were performed using GraphPad Prism 8 (GraphPad, San Diego, CA, USA). Unpaired or paired two-sided Student’s t-tests were used to calculate differences between two groups. one-way or two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test were applied to compare multiple groups. Pearson correlation coefficient test was used to analyze the relationship between variables. Bars represent mean ± SD. P < 0.05 was considered significant. All experiments were carried out at least three times.

Results

WNT4 suppresses crypt regeneration in organoids

Previous study reported that the formation of new branches in organoid mimics the process of crypt fission in tissue [12, 13], therefore, we used organoid culture model to examine the effect of WNT4 on crypt fission. The results showed that knockdown of Wnt4 promoted the branching of organoids, whereas overexpression of Wnt4 suppressed the branch formation of organoids (Fig. 1A–D). Moreover, the number of Ki67-positive cells increased in Wnt4-knockdown organoids while decreased in Wnt4-overexpressing organoids, in both the bud and non-bud areas, compared with their respective controls, although the non-bud areas did not reach statistical significance (Fig. 1E, F), suggesting that WNT4 inhibits the proliferation of organoids. Furthermore, RT-qPCR analysis showed that the expression levels of ISC markers Lgr5, Ascl2 and Olfm4 were increased in Wnt4-knockdown organoids while decreased in Wnt4-overexpressing organoids, compared with their respective controls (Fig. 1G). We also examined the effect of WNT4 on cell differentiation. The results showed that knockdown of Wnt4 significantly upregulated, while overexpression of Wnt4 downregulated the expression of Lyz, Muc2 and Chga. Although Wnt4 knockdown did not significantly alter the levels of Fabp1 and Fabp2, Wnt4 overexpression markedly increased the expression of Fabp1 and Fabp2, suggesting that WNT4 might inhibit the differentiation of secretory epithelial cells but promote the differentiation of absorptive cells (Fig. 1H). Collectively, these results indicate that WNT4 suppresses crypt regeneration via inhibiting crypt fission, ISC activity, cell proliferation, and differentiation.

Fig. 1
figure 1

WNT4 suppresses intestinal organoid branching. A RT-qPCR analysis of the interference efficiency of Wnt4 by siRNA in mouse small intestinal organoids. B Representative images (left) and budding numbers (right) of mouse small intestinal organoids transfected with either control siRNA (si-control) or Wnt4 siRNA (si-Wnt4) after 60 h. Scale bars, 200 μm. C RT-qPCR analysis of the overexpression efficiency of Wnt4 in mouse small intestinal organoids. D Representative images (left) and budding numbers (right) of mouse small intestinal organoids transfected with either vector control (vector) or Wnt4-overexpressing construct (Wnt4-OE) after 60 h. Scale bars, 200 μm. E Representative images of IF staining for Ki67 in mouse small intestinal organoids transfected with either control siRNA or Wnt4 siRNA. Scale bars, 50 μm. The percentages of Ki67-positive cells in the bud and non-bud areas of each organoid (n = 17 for si-control and n = 21 for si-Wnt4) were quantified separately. F Representative images of IF staining for Ki67 in mouse small intestinal organoids transfected with either vector control or Wnt4-overexpressing construct. Scale bars, 50 μm. The percentages of Ki67-positive cells in the bud and non-bud areas of each organoid (n = 16 for both Vector and Wnt4-OE) were quantified separately. G RT-qPCR analysis of the mRNA levels of stem cell marker genes (Lgr5, Ascl2 and Olfm4) in mouse small intestinal organoids after knockdown or overexpression of Wnt4. H RT-qPCR analysis of the mRNA levels of cell differentiation markers (Lyz, Muc2, Chga, Fabp1 and Fabp2) in mouse small intestinal organoids after knockdown or overexpression of Wnt4. All values are means ± SD. Statistical analyses were performed by unpaired Student’s t-test (A–H). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Full size image

WNT4 promotes symmetric fission of crypt in organoids

We further investigated whether WNT4 regulates the symmetry of crypt fission. After transfection of Wnt4 siRNA or overexpression vector, we dynamically monitored the organoid fission process using time-lapse imaging (Fig. 2A, B). Since Eph/Ephrin signaling controls the localization of Paneth cells, which in turn determines the initiation site of crypt fission, the inclusion of inhibitory Eph fragments could lead to more asymmetric fissions. Therefore, we took Eph-treated organoids as positive controls for asymmetric fission. To more accurately analyze the effect of WNT4 on the symmetry of the crypt fission, we compared the length of the two daughter crypts on the basis of 3D structure of the organoids stained against F-actin and nuclei being visualized using confocal microscopy (Fig. 2C, D). We found that the frequency of asymmetric fission increased after suppressing Wnt4 expression (Fig. 2E). Conversely, overexpression of Wnt4 increased the symmetric fissions of the organoids compared with the vector control (Fig. 2F). These results indicate that WNT4 promotes symmetric crypt fission in organoids.

Fig. 2
figure 2

WNT4 promotes symmetric fission of crypt in organoids. A, B Representative time-lapse images of mouse small intestinal organoids transfected with si-control, si-Wnt4, Vector or Wnt4-OE, or treated with an inhibitory Eph fragment (Eph, 10 μg/ml) at the indicated time points. Scale bars, 100 μm. Red dashed boxes indicate symmetric fissions, and yellow dashed boxes indicate asymmetric fissions. C, D Representative 3D projections of organoids stained against F-actin (Phalloidin, red) and nuclei (Hoechst, blue) after transfection with si-control, si-Wnt4, Vector or Wnt4-OE, or treated with an inhibitory Eph fragment (Eph, 10 μg/ml). Scale bars, 50 μm. E, F The symmetry ratio was calculated by dividing the length of the short daughter crypt by the length of the long daughter crypt based on the 3D projections of organoids in C and D. n = 24 for si-control, n = 29 for si-Wnt4, n = 29 for Eph, n = 27 for vector, and n = 20 for Wnt4-OE. All values are means ± SD. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test (E) or unpaired Student’s t-test (F). **p < 0.01, ****p < 0.0001

Full size image

WNT4 promotes the correct localization of Paneth cells at the crypt base

The localization of Paneth cells can affect the symmetry of crypt fission [12]. We next examined whether WNT4 expression affects Paneth cell localization. The 3D projections of organoids stained against lysozyme to visualize Paneth cells revealed that, compared with the control siRNA, knockdown of Wnt4 expression resulted in more Paneth cells being mispositioned away from the crypt base (Fig. 3A, C). Meanwhile, overexpression of Wnt4 led more Paneth cells to localization near the crypt base (Fig. 3B, D). In addition, WNT4 also reduced the total number of Paneth cells in organoid (Additional file 1: Supplementary Fig. 1), in line with the result mentioned above that WNT4 inhibited LYZ expression (Fig. 1H). Collectively, these results suggest that WNT4 may influence the symmetric fission of crypts by regulating the localization of Paneth cells.

Fig. 3
figure 3

WNT4 promotes the correct localization of Paneth cells in the crypt. A, B 3D projections of mouse small intestinal organoids stained to visualize Paneth cells (lysozyme, green), F-actin (Phalloidin, red), and nuclei (Hoechst, blue). Organoids were transfected with si-control or si-Wnt4, or treated with an inhibitory Eph fragment (Eph, 10 μg/ml) in A, or transfected with Vector or Wnt4-OE in B. Scale bars, 25 μm. White arrows indicate Paneth cells positioned in the crypt base and yellow arrows indicate Paneth cells positioned away from the crypt base. C, D The frequency of Paneth cells distanced from the crypt base was quantified on the basis of the 3D projections of organoids in A and B. The tip of the organoid bud was identified as the crypt base. n = 79 Paneth cells for si-control, n = 98 Paneth cells for si-Wnt4, n = 101 Paneth cells for Eph, n = 54 Paneth cells for Vector, and n = 52 Paneth cells for Wnt4-OE

Full size image

WNT4 negatively regulates canonic WNT/β-catenin pathway and depends on ROR2 to promote symmetric fission

Previous studies have reported that β-catenin/TCF signaling regulates the localization of epithelial cells by controlling the expression of EphB/EphrinB [5]. Therefore, we further investigated whether WNT4 mediates the canonic WNT/β-catenin signaling pathway. The results showed that knockdown of Wnt4 significantly increased, while overexpression of Wnt4 decreased the mRNA levels of β-catenin target genes Axin2 and c-Myc in the organoids (Fig. 4A). Moreover, knockdown of Wnt4 also up-regulated, while overexpression of Wnt4 suppressed the mRNA level of Ephb3 in the organoids (Fig. 4A). These data suggest that WNT4 negatively regulates the canonic WNT/β-catenin signaling pathway and EphB3 expression.

Fig. 4
figure 4

WNT4 negatively regulates WNT/β-catenin signaling, and relies on ROR2 receptor to promote symmetric crypt fission. A RT-qPCR analysis of the expression changes of WNT/β-catenin target genes (Axin2, Myc, Ccnd1 and Ephb3) in mouse small intestinal organoids after knockdown (left) or overexpression (right) of Wnt4. B Colocalized expression of ROR2 (green) and WNT4 (red) in both normal mouse small intestinal tissue and organoid was detected by IF staining. Boxed regions are magnified and shown in the right column. Nuclei were counterstained with DAPI (blue). White arrows indicate colocalization. Scale bars, 100 μm (columns 1–4), 50 μm (column 5). C Lysate from the whole mouse small intestine was immunoprecipitated with anti-ROR2 or anti-IgG and analyzed by western blot with anti-WNT4 and anti-ROR2 antibodies. Yellow asterisk indicates the WNT4 band. D The symmetry ratio was calculated by dividing the length of the short daughter crypt by the length of the long daughter crypt on the basis of the 3D projections of mouse small intestinal organoids with overexpression of Wnt4 combined with or without knockdown of Ror2 (n = 20 for vector, n = 22 for Wnt4-OE, n = 24 for Wnt4-OE + si-Ctr, n = 21 for Wnt4-OE + si-Ror2). E The symmetry ratio was calculated by dividing the length of the short daughter crypt by the length of the long daughter crypt on the basis of the 3D projections of mouse small intestinal organoids with either knockdown of Wnt4 or knockdown of Ror2 (n = 19 for si-control, n = 21 for si-Wnt4, n = 20 for si-Ror2). All values are means ± SD. Statistical analyses were performed by unpaired Student’s t-test between two groups (A), and one-way ANOVA with Tukey’s multiple comparisons test (D, E). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Full size image

Since WNT5a inhibits canonic WNT/β-catenin signaling through receptor tyrosine kinase-like orphan receptor ROR2 [30, 31], we hypothesized that WNT4 might also function through ROR2. IF staining showed that WNT4 and ROR2 were both positively expressed in the crypt and co-localized in the epithelial cells in both normal mouse small intestinal tissue and organoid (Fig. 4B). In addition, co-IP assay showed that WNT4 was able to bind to ROR2 in mouse normal small intestine (Fig. 4C). We further investigated whether WNT4 regulates the symmetry of crypt fission depending on ROR2. The results showed that inhibition of Ror2 expression greatly abrogated the promoting effect of Wnt4 overexpression on the symmetric fission of crypt in organoids (Fig. 4D, Additional file 1: Supplementary Fig. 2). Moreover, knockdown of Ror2 had a similar effect as knockdown of Wnt4 in promoting asymmetric crypt fission of organoids (Fig. 4E). These data suggest that WNT4 promotes symmetric fission depending on ROR2.

WNT4 expression is decreased in the radiation-injured intestinal tissue

Through histological examination of RE specimens of patients and animal models, we found that compared with normal intestinal crypts, which were closely arranged in “rows of test tubes” perpendicular to the muscularis mucosae, the regenerated crypts in the radiation-injured intestine usually showed irregular morphology and size, which we speculate may be partly the result of asymmetric fission of crypts during regeneration (Fig. 5A, B). Meanwhile, lysozyme-positive Paneth cells were confined to the base of crypts and colocalized with EphB3 in the mouse normal small intestine; while in the irradiation group, lysozyme-positive and EphB3-positive areas exceeded the base of crypts and expanded upward, which means that Paneth cells were mislocalized and EphB3 expression was increased (Fig. 5C). Furthermore, ISH analysis showed that Wnt4 expression was positive in the crypt and villus mesenchyme of the normal mice, but was decreased in both the crypt and villus mesenchyme of the irradiated mice (Fig. 5D, E). In addition, IHC staining and RT-qPCR analysis further confirmed that WNT4 expression was decreased in the irradiated mouse small intestine, compared with the normal control (Additional file 1: Supplementary Fig. 3A, B). We further examined WNT4 expression in human RE specimens. RT-qPCR analysis showed that WNT4 expression was lower in the lesions of RE than in their paired adjacent non-injured intestinal tissues (Fig. 5F). Moreover, WNT4 expression was negatively correlated with EphB3 expression in human RE (Fig. 5G). Taken together, these results support the hypothesis that decreased expression of WNT4 causes excessive expression of EphB3, which leads to the misposition of Paneth cells and thus the asymmetric fission of crypts during radiation-induced crypt regeneration.

Fig. 5
figure 5

WNT4 expression is decreased in the radiation-injured human and mouse intestinal tissues. A HE staining of the small intestines of normal control mouse (Normal) and mouse with 10.5 Gy of abdominal irradiation after 4 weeks (IR). Scale bars, 100 μm. B HE staining of the radiation-injured lesion (L) and paired adjacent non-injured intestinal tissue (N) in RE patient. Scale bars, 100 μm. C IF staining was performed using anti-lysozyme (labeled Paneth cells, red) and anti-EphB3 (green) antibodies in the small intestinal tissues of normal and IR mice. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. D ISH analysis of Wnt4 expression in the small intestinal tissues of normal and IR mice. The crypt regions (red boxes) were magnified in the middle column, and the villous regions (black boxes) were magnified in the right column. Scale bars, 200 μm (left column) and 50 μm (middle and right columns). E The intensity of Wnt4 expression in the crypts and villus mesenchyme in the normal (n = 6) and IR (n = 6) mice was quantified using ImageJ software based on the ISH analysis in D. F RT-qPCR analysis of WNT4 expression in seven clinical samples of RE lesions (L) and paired adjacent non-injured intestinal tissues (N). G Correlation analysis between the relative WNT4 mRNA level and EphB3 mRNA level in the intestinal lesions of patients with RE (n = 18). Data are means ± SD. Statistical analysis was performed by unpaired Student’s t-test (E), paired Student’s t-test (F), and Pearson correlation coefficient test (G). ns, not significant, *p < 0.05, **p < 0.01

Full size image

WNT4 supplementation mediated by AAV promotes the symmetric fission of crypts after irradiation in mice

We next explored whether Wnt4 overexpression delivered by AAV could promote more symmetric fission of the crypts in a mouse radiation-injury model. AAV-Wnt4 or AAV-vector was injected intraperitoneally the following day after 10.5 Gy of abdominal irradiation. At 4 weeks after irradiation, crypt regeneration in small intestine was assessed. IF staining of anti-Flag showed that WNT4 was successfully overexpressed in the small intestinal epithelium in the irradiated AAV-Wnt4-treated group (Fig. 6A). RT-qPCR analysis also showed that, compared with the sham-irradiated control group, Wnt4 mRNA expression was significantly decreased in the small intestine of the irradiated AAV-vector-treated mice, while it was greatly restored in the irradiated AAV-Wnt4 group (Fig. 6B). IF staining showed that, compared with the sham-irradiated control group, the intensity of EphB3 expression and the height of the EphB3-positive area were increased in the irradiated AAV-vector group, whereas these were largely decreased in the irradiated AAV-Wnt4 group (Fig. 6C, D). Furthermore, in the irradiated AAV-vector group, more Paneth cells were located far from the crypt base, whereas in the irradiated AAV-Wnt4 group, more Paneth cells were distributed near the crypt base (Fig. 6C, E).

Fig. 6
figure 6

AAV-mediated WNT4 supplementation inhibits EphB3 expression in small intestinal crypts in mice with radiation injury. The mice received 10.5 Gy of abdominal irradiation or sham-irradiation as control. The next day, AAV-vector or AAV-Wnt4 was intraperitoneally injected into the irradiated mice. At 4 weeks after irradiation, the small intestinal tissues were collected and analyzed. A The overexpression of WNT4 in the small intestines of irradiated mice treated with AAV-Wnt4 was confirmed by IF staining with anti-Flag antibody (green). Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. Dashed lines indicate crypt area. a: apical; b: basal. B RT-qPCR analysis of Wnt4 expression in the entire small intestinal tissues of the irradiated mice treated with AAV-vector (n = 5) or AAV-Wnt4 (n = 5) or the sham-irradiated control mice (n = 3). C IF staining was performed using anti-lysozyme (labeled Paneth cells, red) and anti-EphB3 (green) antibodies in the small intestinal tissues of the three groups of mice. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. Dashed lines indicate crypt area. a: apical; b: basal. D The intensity of EphB3 expression and the height of the EphB3-positive area were quantified using ImageJ on the basis of the IF staining of EphB3 in C. E The frequency of Paneth cells distanced from the crypt base was quantified using ImageJ on the basis of the IF staining of lysozyme in C. Values are means ± SD. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test (B, D). *p < 0.05, **p < 0.01, ***p < 0.001

Full size image

We further calculated the proportions of crypts with symmetric or asymmetric fission in the small intestines in each treatment groups on the basis of HE staining of tissue sections (Fig. 7A). Our results showed that symmetric fission occasionally occurred and asymmetric fission was much rarer in normal mouse small intestine (Fig. 7A), which is similar to the findings in colon [32, 33]. However, in the irradiated AAV-vector-treated mice, asymmetric fission was significantly increased (Fig. 7A), which further supports our previous observation that the size and morphology of the regenerated crypts were irregular after irradiation (Fig. 5A, B). Importantly, restoration of WNT4 expression after irradiation in AAV-Wnt4-treated mice resulted in a significant decrease in asymmetric fission, while a concomitant increase in symmetric fission was observed (Fig. 7A). Moreover, IHC staining showed that the number of mislocated Paneth cells was significantly increased in the small intestines of irradiated mice with AAV-vector treatment compared with the sham-irradiated control mice, while it was decreased in the irradiated AAV-Wnt4-treated mice (Fig. 7B). In addition, there was no significant difference in the number of Ki67-positive cells per crypt among these groups (Fig. 7B). Taken together, these results suggest that restoration of WNT4 expression could promote the correct localization of Paneth cells, reduce asymmetric fission of crypts, and ultimately improve the regularity of the reconstructed intestinal epithelium after radiation injury.

Fig. 7
figure 7

AAV-mediated WNT4 supplementation reduces asymmetric fission of small intestinal crypts in mice with radiation injury. A Representative images of HE staining (left) of the small intestinal tissues of the irradiated mice treated with AAV-vector (n = 5) or AAV-Wnt4 (n = 5) or the sham-irradiated control mice (n = 3). The proportions of symmetric and asymmetric crypt fissions in the small intestinal tissues of mice in the three groups were calculated (right). The black and red arrow heads in the sham-irradiated control group indicate crypt not undergoing fission and crypt undergoing symmetric fission, respectively, which are magnified in the next row (left). The yellow arrows in the irradiated AAV-vector group indicate crypts undergoing asymmetric fissions, which are magnified in the next row (middle). The red arrows in the irradiated AAV-Wnt4 group indicate crypts undergoing symmetric fissions, which are magnified in the next row (right). Scale bars, 50 μm (upper row) and 25 μm (lower row). B Representative images of lysozyme and Ki67 staining of the small intestinal tissues of the irradiated mice treated with AAV-vector (n = 5) or AAV-Wnt4 (n = 5) or the sham-irradiated control mice (n = 3). Scale bars, 50 μm. The numbers of mislocated Paneth cells per 20 × field and Ki67+ cells per crypt were quantified. All values are means ± SD. Statistical analyses were performed by two-way ANOVA (A) or one-way ANOVA (B) with Tukey’s multiple comparisons test. ns, not significant. *p < 0.05, **p < 0.01, ****p < 0.0001

Full size image

Discussion

In intestinal tissues after radiation injury, it is often observed that the size and shape of the regenerated crypts are irregular, disordered, and the normal arrangement pattern of the intestinal epithelium is disrupted, which may be related to excessive asymmetric fission of crypts during regeneration. Asymmetric crypt fission that occurs in inflammatory bowel disease is considered to be a kind of pathological aberration with a tendency to develop mucosal dysplasia [32]. However, little is known about the mechanisms regulating the symmetry of crypt fission. In this study, we found that WNT4 expression is inhibited in the intestinal tissue after radiation injury. Using organoid culture model, we further discovered that WNT4 promotes the symmetric fission of crypts by regulating the canonic WNT/β-catenin signaling pathway. Importantly, WNT4 supplementation effectively promotes more symmetric crypt fissions in a radiation-injury animal model, improving the regularity of the reconstructed intestinal epithelium.

Although it was reported that the incidence of symmetric and asymmetric fission is similar during normal intestinal development [12], it seems that the regeneration of crypts after radiation injury is prone to more asymmetric fissions, which may be partially owing to low crypt density, high fission frequency, and uneven mechanical pressure from the surrounding environment. Indeed, in colorectal adenomas and ulcerative colitis, a high frequency of crypt fission events is often accompanied by a higher rate of asymmetric fission, whereas in normal adult colonic mucosa, crypt fission rarely occurs [9, 11, 32, 34, 35]. However, on the basis of our findings, we propose that another important factor contributing to the increased asymmetric fission is the altered expression of certain key molecules that regulate crypt fission, such as decreased expression of WNT4. Using the organoid culture system and an animal model of RE, our study provides evidence that decreased WNT4 is responsible for increased EphB3, mislocalized Paneth cells, and excessive asymmetric crypt fission in the radiation-injured small intestine. A high proportion of asymmetric and irregular crypts will cause the intestinal mucosa to lose its normal morphological structure, which may cause the disorder of intestinal function.

The Eph receptor tyrosine kinases mediate cell repulsion and compartmentalization during embryonic development [36, 37]. When engaged with their ephrin ligands, the bidirectional signal is conveyed in both the Eph-expressing (forward signaling) and the ephrin-expressing (reverse signaling) cells [38]. In the intestinal epithelium, EphB/ephrinB signaling dictates cell positioning and ordered migration [5]. In the adult normal intestine, EphB and ephrinB expression are controlled by β-catenin signaling, and their expression patterns show inverse gradients along the crypt-villus axis, that is, high expression of EphB3 is restricted to the crypt base, while ephrinB1 expression is strongly detected at the crypt-villus junction and gradually decreases toward the crypt bottom [5]. EphB3 deletion or blocking EphB3 signaling results in Paneth cells not being located at the crypt base, but randomly spreading throughout the crypt [5, 6]. Alternatively, the upregulation of EphB3 expression owing to constitutive β-catenin signaling activation also leads to mislocalization of Paneth cells [39,40,41], suggesting that disrupting the expression gradient of EphB3 could impair the directed migration of Paneth cells. Our study demonstrated that the decreased expression of WNT4 in radiation-injuried intestinal epithelium leads to overactivation of β-catenin pathway and increase of EphB3, with EphB3 not being confined to the crypt base, but expanding upward, thus causing the misposition of Paneth cells. This was further supported by our in vivo evidence that WNT4 supplementation promotes restoration of the normal gradient of EphB3 and proper localization of Paneth cells in the animal model.

WNT ligands can selectively activate either the canonic β-catenin signaling or the non-canonic pathways largely depending on the availability of their receptors. For example, WNT5a can activate the non-canonic JNK signaling and inhibit canonic WNT/β-catenin signaling through ROR2 [30, 31, 42, 43]; on the contrary, WNT5a amplified canonic β-catenin signaling when the Frizzled (Fzd) receptor Fzd4 is expressed [31]. Studies have demonstrated that WNT4 could regulate the canonic β-catenin signaling and non-canonic PCP/JNK and Ca2+/CaMK pathways via interacting with Fzds [23, 44, 45]. However, whether WNT4 relies on ROR receptors to activate downstream signaling in regulating certain cellular processes is rarely reported. Our study demonstrated that ROR2 is expressed in the crypt epithelial cells and interacts with WNT4. Moreover, disrupting the expression of ROR2 eliminates the promotion effect of WNT4 on symmetric fission of crypt, suggesting that ROR2 might be a bona-fide functional receptor of WNT4.

Conclusions

Our study demonstrates that WNT4 suppresses the expression of EphB3, which is elevated in RE, through inhibition of β-catenin pathway, thereby promoting the correct localization of Paneth cells as well as the symmetric fission of small intestinal crypts. Overexpression of WNT4 effectively promotes the symmetric fission of small intestinal crypts and thus improves the regularity of the regenerating crypts in mice with radiation-induced injury, suggesting that WNT4 could be an attractive therapeutic target for RE.

Availability of data and materials

The data supporting this study are available from the corresponding author upon reasonable request.

Abbreviations

ISCs:

Intestinal stem cells

AAV:

Adeno-associated virus

RE:

Radiation enteritis

PCP:

Planar cell polarity

IHC:

Immunohistochemistry

IF:

Immunofluorescence

HE:

Hematoxylin and eosin

IP:

Immunoprecipitation

ISH:

In situ hybridization

Fzd:

Frizzled

References

  1. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449:1003–7.

    Article  CAS  PubMed  Google Scholar 

  2. van der Flier LG, Clevers H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol. 2009;71:241–60.

    Article  PubMed  Google Scholar 

  3. Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol. 2014;15:19–33.

    Article  CAS  PubMed  Google Scholar 

  4. Stappenbeck TS, Wong MH, Saam JR, Mysorekar IU, Gordon JI. Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium. Curr Opin Cell Biol. 1998;10:702–9.

    Article  CAS  PubMed  Google Scholar 

  5. Batlle E, Henderson JT, Beghtel H, van den Born MM, Sancho E, Huls G, et al. Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell. 2002;111:251–63.

    Article  CAS  PubMed  Google Scholar 

  6. Holmberg J, Genander M, Halford MM, Anneren C, Sondell M, Chumley MJ, et al. EphB receptors coordinate migration and proliferation in the intestinal stem cell niche. Cell. 2006;125:1151–63.

    Article  CAS  PubMed  Google Scholar 

  7. Clarke RM. The effect of growth and of fasting on the number of villi and crypts in the small intestine of the albino rat. J Anat. 1972;112:27–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. St Clair WH, Osborne JW. Crypt fission and crypt number in the small and large bowel of postnatal rats. Cell Tissue Kinet. 1985;18:255–62.

    CAS  PubMed  Google Scholar 

  9. Wasan HS, Park HS, Liu KC, Mandir NK, Winnett A, Sasieni P, et al. APC in the regulation of intestinal crypt fission. J Pathol. 1998;185:246–55.

    Article  CAS  PubMed  Google Scholar 

  10. Totafurno J, Bjerknes M, Cheng H. The crypt cycle. Crypt and villus production in the adult intestinal epithelium. Biophys J. 1987;52:279–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tan CW, Hirokawa Y, Gardiner BS, Smith DW, Burgess AW. Colon cryptogenesis: asymmetric budding. PLoS ONE. 2013;8: e78519.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Langlands AJ, Almet AA, Appleton PL, Newton IP, Osborne JM, Nathke IS. Paneth cell-rich regions separated by a cluster of Lgr5+ cells initiate crypt fission in the intestinal stem cell niche. PLoS Biol. 2016;14: e1002491.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459:262–5.

    Article  CAS  PubMed  Google Scholar 

  14. Clevers HC, Bevins CL. Paneth cells: maestros of the small intestinal crypts. Annu Rev Physiol. 2013;75:289–311.

    Article  CAS  PubMed  Google Scholar 

  15. Pin C, Parker A, Gunning AP, Ohta Y, Johnson IT, Carding SR, et al. An individual based computational model of intestinal crypt fission and its application to predicting unrestrictive growth of the intestinal epithelium. Integr Biol. 2015;7:213–28.

    Article  Google Scholar 

  16. Gregorieff A, Clevers H. Wnt signaling in the intestinal epithelium: from endoderm to cancer. Genes Dev. 2005;19:877–90.

    Article  CAS  PubMed  Google Scholar 

  17. Mah AT, Yan KS, Kuo CJ. Wnt pathway regulation of intestinal stem cells. J Physiol. 2016;594:4837–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kuhnert F, Davis CR, Wang HT, Chu P, Lee M, Yuan J, et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc Natl Acad Sci USA. 2004;101:266–71.

    Article  CAS  PubMed  Google Scholar 

  19. Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet. 2006;7:349–59.

    Article  CAS  PubMed  Google Scholar 

  20. Fevr T, Robine S, Louvard D, Huelsken J. Wnt/beta-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells. Mol Cell Biol. 2007;27:7551–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Veeman MT, Axelrod JD, Moon RT. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell. 2003;5:367–77.

    Article  CAS  PubMed  Google Scholar 

  22. Miyoshi H, Ajima R, Luo CT, Yamaguchi TP, Stappenbeck TS. Wnt5a potentiates TGF-beta signaling to promote colonic crypt regeneration after tissue injury. Science. 2012;338:108–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang Q, Pan Y, Ji J, Xu Y, Zhang Q, Qin L. Roles and action mechanisms of WNT4 in cell differentiation and human diseases: a review. Cell Death Discov. 2021;7:287.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hauer-Jensen M, Denham JW, Andreyev HJ. Radiation enteropathy–pathogenesis, treatment and prevention. Nat Rev Gastroenterol Hepatol. 2014;11:470–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cairnie AB, Millen BH. Fission of crypts in the small intestine of the irradiated mouse. Cell Tissue Kinet. 1975;8:189–96.

    CAS  PubMed  Google Scholar 

  26. Wu P, Li L, Wang H, Ma T, Wu H, Fan X, et al. Role of angiogenesis in chronic radiation proctitis: new evidence favoring inhibition of angiogenesis ex vivo. Dig Dis Sci. 2018;63:113–25.

    Article  CAS  PubMed  Google Scholar 

  27. Rubio CA, Schmidt PT. Asymmetric crypt fission in sessile serrated lesions. J Clin Pathol. 2021;74:712–7.

    Article  CAS  PubMed  Google Scholar 

  28. Cui Y, Wu H, Liu Z, Ma T, Liang W, Zeng Q, et al. CXCL16 inhibits epithelial regeneration and promotes fibrosis during the progression of radiation enteritis. J Pathol. 2023;259:180–93.

    Article  CAS  PubMed  Google Scholar 

  29. Morgan RG, Chambers AC, Legge DN, Coles SJ, Greenhough A, Williams AC. Optimized delivery of siRNA into 3D tumor spheroid cultures in situ. Sci Rep. 2018;8:7952.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. van Amerongen R, Fuerer C, Mizutani M, Nusse R. Wnt5a can both activate and repress Wnt/beta-catenin signaling during mouse embryonic development. Dev Biol. 2012;369:101–14.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Mikels AJ, Nusse R. Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol. 2006;4: e115.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Rubio CA, Schmidt PT. Asymmetric crypt fission in colectomy specimens in patients with ulcerative colitis. J Clin Pathol. 2021;74:577–81.

    Article  PubMed  Google Scholar 

  33. Baker AM, Cereser B, Melton S, Fletcher AG, Rodriguez-Justo M, Tadrous PJ, et al. Quantification of crypt and stem cell evolution in the normal and neoplastic human colon. Cell Rep. 2019;27:2524.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wong WM, Mandir N, Goodlad RA, Wong BC, Garcia SB, Lam SK, et al. Histogenesis of human colorectal adenomas and hyperplastic polyps: the role of cell proliferation and crypt fission. Gut. 2002;50:212–7.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Rubio CA, Schmidt PT. Morphological classification of corrupted colonic crypts in ulcerative colitis. Anticancer Res. 2018;38:2253–9.

    PubMed  Google Scholar 

  36. Pasquale EB. Eph receptor signalling casts a wide net on cell behaviour. Nat Rev Mol Cell Biol. 2005;6:462–75.

    Article  CAS  PubMed  Google Scholar 

  37. Poliakov A, Cotrina M, Wilkinson DG. Diverse roles of Eph receptors and ephrins in the regulation of cell migration and tissue assembly. Dev Cell. 2004;7:465–80.

    Article  CAS  PubMed  Google Scholar 

  38. Himanen JP, Rajashankar KR, Lackmann M, Cowan CA, Henkemeyer M, Nikolov DB. Crystal structure of an Eph receptor-ephrin complex. Nature. 2001;414:933–8.

    Article  CAS  PubMed  Google Scholar 

  39. Sansom OJ, Reed KR, Hayes AJ, Ireland H, Brinkmann H, Newton IP, et al. Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev. 2004;18:1385–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. van Es JH, Jay P, Gregorieff A, van Gijn ME, Jonkheer S, Hatzis P, et al. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nat Cell Biol. 2005;7:381–6.

    Article  PubMed  Google Scholar 

  41. Dominguez-Brauer C, Hao Z, Elia AJ, Fortin JM, Nechanitzky R, Brauer PM, et al. Mule regulates the intestinal stem cell niche via the Wnt pathway and targets EphB3 for proteasomal and lysosomal degradation. Cell Stem Cell. 2016;19:205–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, et al. The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway. Genes Cells. 2003;8:645–54.

    Article  CAS  PubMed  Google Scholar 

  43. Maeda K, Kobayashi Y, Udagawa N, Uehara S, Ishihara A, Mizoguchi T, et al. Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis. Nat Med. 2012;18:405–12.

    Article  CAS  PubMed  Google Scholar 

  44. Li X, Li Z, Wang J, Li Z, Cui H, Dai G, et al. Wnt4 signaling mediates protective effects of melatonin on new bone formation in an inflammatory environment. FASEB J. 2019;33:10126–39.

    Article  CAS  PubMed  Google Scholar 

  45. Shukla V, Kaushal JB, Kumar R, Popli P, Agnihotri PK, Mitra K, et al. Microtubule depolymerization attenuates WNT4/CaMKIIalpha signaling in mouse uterus and leads to implantation failure. Reproduction. 2019;158:47–59.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Xiaoli Hu and Jin Li from Leica Microsystems Trading Ltd Guangzhou Branch for their technical assistance in the 3D reconstruction of organoids. We also thank the support of National Key Clinical Discipline of China.

Funding

This work was supported by The Natural Science Foundation of Guangdong Province (2021A1515010080 to Y.C.).

Author information

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, Guangdong Institute of Gastroenterology, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of China

    Jingyang Cheng, Haiyong Wu & Yanmei Cui

  2. Biomedical Innovation Center, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of China

    Jingyang Cheng, Haiyong Wu & Yanmei Cui

Authors
  1. Jingyang Cheng
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Haiyong Wu
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Yanmei Cui
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Y.C. designed and supervised the study; J.C. and H.W. carried out experiments; Y.C. and J.C. wrote the manuscript. All authors were involved in revising the paper and approved the final version of the paper.

Corresponding author

Correspondence to Yanmei Cui.

Ethics declarations

Ethics approval and consent to participate

All studies involving the use of human specimens were approved by the institutional review board of the Sixth Affiliated Hospital, Sun Yat-sen University (approval no. 2024ZSLYEC-522, date: 26 September 2024), and performed in accordance with the principles of the Declaration of Helsinki. Prior written consent from all subjects was obtained. The animal study was approved by the institutional animal care and use committee (IACUC) of Ruige Biotechnology (approval no. 20231031-004, date: 31 October 2023), and performed in accordance with the Basel Declaration.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

11658_2024_677_MOESM1_ESM.docx

Additional file 1: Supplementary Fig. 1. WNT4 inhibits the differentiation of Paneth cells. Supplementary Fig. 2. WNT4 promotion of symmetric crypt fission is dependent on the ROR2 receptor. Supplementary Fig. 3. WNT4 expression is decreased in radiation-injured intestinal tissues of mice. Supplementary Table 1. Primer sequences for RT-qPCR.

Additional file 2. Raw images of IP.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cheng, J., Wu, H. & Cui, Y. WNT4 promotes the symmetric fission of crypt in radiation-induced intestinal epithelial regeneration. Cell Mol Biol Lett 29, 158 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s11658-024-00677-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s11658-024-00677-4

Keywords

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us