Wnt5a potentiates TGF-β signaling to promote colonic crypt regeneration after tissue injury

Abstract

Reestablishing homeostasis after tissue damage depends on the proper organization of stem cells and their progeny, though the repair mechanisms are unclear. The mammalian intestinal epithelium is well suited to approach this problem, as it is composed of well-delineated units called crypts of Lieberkühn. We found that Wnt5a, a noncanonical Wnt ligand, was required for crypt regeneration after injury in mice. Unlike controls, Wnt5a-deficient mice maintained an expanded population of proliferative epithelial cells in the wound. We used an in vitro system to enrich for intestinal epithelial stem cells to discover that Wnt5a inhibited proliferation of these cells. Surprisingly, the effects of Wnt5a were mediated by activation of transforming growth factor-β (TGF-β) signaling. These findings suggest a Wnt5a-dependent mechanism for forming new crypt units to reestablish homeostasis.

Fig. 1

Colonic crypts regenerate from existing crypts during injury repair. (A) An H&E–stained section of a wound at day 6 post-injury. An asterisk indicates the center of the wound bed. An arrowhead indicates a wound channel that consists of immature epithelial cells (boxed region, see inset). Crypts distant from the wound site are represented in the panel labeled ‘Uninjured area’. Bar, 200 μm. (B) Sections stained for Ki-67 (brown) to label proliferative cells at various time points after biopsy injury. An arrowhead labels the wound channel at day 6 post-injury. Bars, 100 μm. n=3 wounds/time point for (A) and (B). (C) Migration of clonal cell populations from labeled crypts after wounding in Vil-CreERT: Rosa26R mice. Cells expressing LacZ were visualized by the staining with 5bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). Dotted lines outline original wound area. Bars, 200 μm. (D) An H&E–stained section of a wound at day 8 post-injury in a Vil-CreERT: Rosa26R mouse. LacZ-positive cells (blue) were present in a wound channel (arrowhead; see inset). The wound bed is delineated by a bracket. Bar, 200 μm. (E) An H&E–stained section of a wound at day 28 post-injury in a Vil-CreERT: Rosa26R mouse shows clusters of LacZ-positive crypts in the wound bed (defined by bracket). Bar, 200 μm. n=6 wounds/time point for (C), (D) and (E).

Fig. 2

Wnt5a-positive mesenchymal cells stimulate crypt regeneration after injury. (A) RT-PCR analysis of Wnt5a from RNAs isolated from an E13.5 embryo and its placenta tissue (controls), the wound bed and adjacent uninjured mucosa. (B) Plots of mean (+SD) relative Wnt5a mRNA expression levels as determined by quantitative RT-PCR analysis of wound beds and adjacent uninjured mucosa at day 4 post-injury. Data were analyzed using Student's t-test (n=4/group). (C) Mouse colon sections at day 6 post-injury (uninjured area and wound) stained by in situ hybridization for Wnt5a (purple) and hyaluronic acid (basement membrane, brown). Dotted lines outline the apical epithelial surface. Bars, 100 μm. (D) Serial sections of uninjured area and a colonic wound at day 6 post-injury stained for Wnt5a and Axin2 mRNA, respectively. Arrowheads indicate Wnt5a-positive cells associated with wound channel clefts (insets). Methyl green labeled nuclei. Bars, 100 μm. (E) Serial sections of a colonic wound channel at day 6 post-injury stained for Wnt5a mRNA (left) and Ki-67 (right). Wnt5a-positive cells were localized near quiescent epithelial cells (arrowheads). Sections were counterstained with nuclear fast red (left) and hematoxylin (right). Bars, 100 μm. n=3 wounds/assay. (F) Sections from a CAGGCreER™:Wnt5a^+/+ (Wnt5a^+/+) mouse and a CAGGCreER^TM:Wnt5a^flox/flox (Wnt5a^ko/ko) mouse at day 6 post-injury stained by H&E. Arrowheads indicate wound channel invaginations. The arrow indicates an immature wound channel without invaginations. Bars, 200 μm. (G) Graph of the average distance between wound channel invaginations (± SD) (n=7/group). Each dot represents the average distance for an individual wound channel. Data were analyzed using Student's t-test. (H) H&E-stained sections from an Ubc-CreERT2:Wnt5a^flox/+ (Wnt5a^ko/+) and Ubc-Cre-ER^T2:Wnt5a^flox/flox (Wnt5a^ko/ko) mouse at day 8 post-injury (n=3 mice analyzed/group). Arrowheads indicate the space between crypt-like structures that developed from wound channels. Arrows indicate abnormal immature wound channels with no crypt-like structures. Bars, 500 μm. (I) Schematic diagrams of defect in crypt regeneration in Wnt5a^ko/ko mice.

Fig. 3

Wnt5a inhibits proliferation of colonic epithelial stem cells. (A) Focal Wnt5a-induced clefts in colonic epithelial organoids. A control (left panel) or a Wnt5a-soaked bead (right panel) was placed adjacent to different colonic organoids. Bars, 200 μm. (B) Plot of the mean cleft incidence of colonic organoids (+ SD) attached to control or Wnt5a-soaked beads (n=3 experiments). A Student's t-test was used to determine significance. (C) Colonic organoids attached to either control (left panel) or Wnt5a-soaked beads (right panel) were stained for Ki-67 (green). Yellow dotted lines outline the bead attachment area. Nuclei were counterstained with bis-benzimide (blue). Representative images from 3 samples (per group) were shown. Bars, 200 μm. (D) Representative images of colonic epithelial organoids cultured for 48 hours in indicated conditions (n=3 experiments). Bars, 500 μm.

Fig. 4

Wnt5a activates TGF-β signaling pathway. (A) Colonic organoids were cultured for 24 hours with recombinant Wnt5a. Plots of mean (+ SD) relative mRNA expression levels of Serpine1 and Mki67 were determined by quantitative RT-PCR analysis (n=3/group). Data were analyzed using one-way ANOVA followed by Tukey's test (P<0.0001). The asterisk indicates differences compared to the baseline condition (0 ng/ml) that were significant (P<0.05) in the post test. (B) Nuclear localization of p-Smad3 protein. Colonic epithelial cells grown on Matrigel coated chambers were incubated without ligands (control) and with either Wnt5a (400 ng/ml) or TGF-β1 (1 ng/ml) for 2 hours and then fixed and stained for p-Smad3 (representative images from three experiments). Bars, 10 μm. (C) Distribution of p-Smad3 in the wound channels (right panels) and uninjured crypt units (left panels). Colonic sections from Ubc-Cre-ER^T2: Wnt5a^+/+ (Wnt5a^+/+) and Ubc-Cre-ER^T2: Wnt5a^flox/flox(Wnt5a^ko/ko) wounds at day 6 post-injury were stained for p-Smad3 (bottom panels). Cell nuclei were visualized with bis-benzimide (top panels). Arrowheads and arrows indicated the base of wound channels. Bars, 50 μm. (D) Quantification of p-Smad3 in wound channels. Plots of the mean ratio of signal intensity (+ SD) in epithelial cells located in the base of wound channels compared to the base of crypts in unwounded areas (from the same tissue section) were determined as described in Methods (n=4 wounds/group). (E) Colonic organoids were cultured for 24 hours without ligands (control) and with Wnt5a (400 ng/ml), SB-431542 (10 μM) or both together. Representative bright field pictures were shown (n=3 experiments). Bars, 500 μm. (F) Colonic organoids were cultured for 24 hours without ligands (control) and with Wnt5a (400 ng/ml) and TGF-β1 (1 ng/ml). Two pairs of populations expressing shRNA for independent target sequences and their controls (SHC002) were examined. Plots of mean (+ SD) relative mRNA expression levels were determined by quantitative RT-PCR analysis (n=3/group). The asterisk in (D) and (F) indicates differences that were significant in the Student's t-test. * *P<0.01, * * *P<0.001, * * * *P<0.0001.