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Science 338 (6103): 108-113

Copyright © 2012 by the American Association for the Advancement of Science

Wnt5a Potentiates TGF-β Signaling to Promote Colonic Crypt Regeneration After Tissue Injury

Hiroyuki Miyoshi1, Rieko Ajima2,*, Christine T. Luo1, Terry P. Yamaguchi2,{dagger}, and Thaddeus S. Stappenbeck1,{dagger}

1 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
2 Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, MD 21702, USA.

Figure 1
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Fig. 1. Colonic crypts regenerate from existing crypts during injury repair. (A) A hematoxylin and eosin (H&E)–stained section of a wound at day 6 postinjury. The asterisk indicates the center of the wound bed; the 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." Scale bars, 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 postinjury. Scale bars, 100 μm. n = 3 wounds per 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 5-bromo-4-chloro-3-indolyl-β-D-galactoside. Dotted lines outline the original wound area. Scale bars, 200 μm. (D) A H&E-stained section of a wound at day 8 postinjury 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. Scale bar, 200 μm. (E) A H&E-stained section of a wound at day 28 postinjury in a Vil-CreERT: Rosa26R mouse shows clusters of LacZ-positive crypts in the wound bed (shown by the bracket). Scale bar, 200 μm. n = 6 wounds per time point for (C), (D), and (E).


Figure 2
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Fig. 2. Wnt5a-positive mesenchymal cells stimulate crypt regeneration after injury. (A) RT-PCR analysis of Wnt5a from RNAs isolated from an embryonic day 13.5 embryo and its placenta tissue (controls), the wound bed, and adjacent uninjured mucosa. Gapdh, glyceraldehyde-3-phosphate dehydrogenase. (B) Plots of mean (+SD; error bar) relative Wnt5a mRNA expression levels as determined by quantitative RT-PCR analysis of wound beds and adjacent uninjured mucosa at day 4 postinjury. Data were analyzed using the Student’s t test (n = 4 wounds per group). (C) Mouse colon sections at day 6 postinjury (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. Scale bars, 100 μm. (D) Serial sections of uninjured area and a colonic wound at day 6 postinjury stained for Wnt5a and Axin2 mRNA, respectively. Arrowheads indicate Wnt5a-positive cells associated with wound-channel clefts (insets). Methyl green labeled nuclei are shown. Scale bars, 100 μm. (E) Serial sections of a colonic wound channel at day 6 postinjury stained for Wnt5a mRNA (top) and Ki-67 (bottom). Wnt5a-positive cells (arrowheads) were localized near quiescent epithelial cells. Sections were counterstained with nuclear fast red (top) and hematoxylin (bottom). Scale bars, 100 μm. n = 3 wounds per assay. (F) Sections from a CAGGCreERTM:Wnt5a+/+ (Wnt5a+/+) mouse and a CAGGCreERTM:Wnt5aflox/flox (Wnt5ako/ko) mouse at day 6 postinjury stained by H&E. Arrowheads indicate wound-channel invaginations. The arrow indicates an immature wound channel without invaginations. Scale bars, 200 μm. (G) Graph of the average distance between wound-channel invaginations (±SD) (n = 7 wounds per 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 a Ubc-Cre-ERT2:Wnt5aflox/+ (Wnt5ako/+) and Ubc-Cre-ERT2:Wnt5aflox/flox (Wnt5ako/ko) mouse at day 8 postinjury (n = 3 mice analyzed per group). Arrowheads indicate the space between cryptlike structures that developed from wound channels. Arrows indicate abnormal immature wound channels with no cryptlike structures. Scale bars, 500 μm. (I) Schematic diagrams of defects in crypt regeneration in Wnt5ako/ko mice.


Figure 3
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Fig. 3. Wnt5a inhibits proliferation of colonic epithelial stem cells. (A) Focal Wnt5a-induced clefts in colonic epithelial organoids. A control (left) or a Wnt5a-soaked bead (right) was placed adjacent to different colonic organoids. Scale bars, 200 μm. (B) Plot of the mean cleft incidence of colonic organoids (+SD; error bars) 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) or Wnt5a-soaked beads (right) were stained for Ki-67 (green). Yellow dotted lines outline the bead attachment area. Nuclei were counterstained with bis-benzimide (blue). Representative images from three samples (per group) are shown. Scale bars, 200 μm. (D) Representative images of colonic epithelial organoids cultured for 48 hours in Wnt3a/Rspo1 and indicated amounts of Wnt5a. The rightmost panel is a control without Wnt3a/Rspo1 (n = 3 experiments). Scale bars, 500 μm.


Figure 4
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Fig. 4. Wnt5a activates the TGF-β signaling pathway. (A) Colonic organoids were cultured for 24 hours with recombinant Wnt5a. Plots of mean (+SD; error bars) relative mRNA expression levels of Serpine1 and Mki67 were determined by quantitative RT-PCR analysis (n = 3 samples per group). Data were analyzed using one-way analysis of variance followed by Tukey’s test (P < 0.0001). Asterisks indicate differences, compared with the baseline condition (0 ng/ml), that were significant (P < 0.05) in the posttest. (B) Nuclear localization of p-Smad3 protein. Colonic epithelial cells grown on Matrigel-coated chambers (Matrigel, BD, Franklin Lakes, NJ) 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). Scale bars, 10 μm. (C) Distribution of p-Smad3 in the wound channels (right) and uninjured crypt units (left). Colonic sections from Ubc-Cre-ERT2: Wnt5a+/+ (Wnt5a+/+) and Ubc-Cre-ERT2: Wnt5aflox/flox (Wnt5ako/ko) wounds at day 6 postinjury were stained for p-Smad3 (bottom). Cell nuclei were visualized with bis-benzimide (top). Arrowheads and arrows indicate the base of wound channels. Scale 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 with the base of crypts in unwounded areas (from the same tissue section) were determined as described in the supplemental materials and methods (n = 4 wounds per 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). DMSO, dimethyl sulfoxide. Scale 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 samples per group). The asterisks in (D) and (F) indicate differences that were significant in the Student’s t test: **P < 0.01, ***P < 0.001, ****P < 0.0001.


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