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Re-establishing 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 non-canonical 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 found that Wnt5a inhibited proliferation of intestinal epithelial stem cells using an in vitro system to enrich for these cells. Surprisingly, the effects of Wnt5a were mediated by activation of transforming growth factor (TGF)-β signaling. These findings suggest a Wnt5a-dependent mechanism for forming new crypt units to re-establish homeostasis.
Tissue regeneration requires proper spatial allocation and organization of new stem cells for efficient return to homeostasis (1, 2). Crypts of Lieberkühn are subunits that house intestinal stem cells and are lost in response to a variety of insults including ischemia, infection, irradiation and inflammatory bowel disease (3). Although individual crypts undergo fission to replicate during homeostasis (fig. S1A) (4, 5), the mechanism of their regeneration is unknown. Thus, crypt regeneration is a proxy for proper stem cell organization and provides an excellent system to uncover new principles underlying stem cell replacement/organization in vivo.
To model crypt/epithelial stem cell loss, we previously developed an injury system to focally excise crypts from the absorptive inner lining of the mouse colon (6). In response to the excision of ~1 mm2 areas from the inner lining of mouse colons (~250-300 crypts), a reproducible program of epithelial alteration occurred. During the first phase (0-4 days post-injury), a flattened layer of non-proliferative epithelial cells (wound-associated epithelial cells; WAE) emanated from crypts adjacent to the wound and migrated over the wound bed surface. During the second phase (4-8 days post-injury), crypts adjacent to the wound formed lateral, open extensions towards the center of the wound bed, forming an array of channel-like structures (fig. S1, B and C). Histologic cross-sections of wound channels at day 6 post-injury showed that they resembled crypts (Fig. 1A) but were distinguished by a predominately proliferative, undifferentiated cell population (Fig. 1B and fig. S1, D and E) (7).
We hypothesized that new crypts within wounds arose from existing crypts adjacent to the wound bed (6). We therefore performed lineage tracing experiments using Vil-CreERT: Gtrosa26tm1Sor (Rosa26R) mice (8, 9). To mark a subset of crypts prior to injury, we activated Cre recombinase in the intestinal epithelium with a single tamoxifen injection followed by a one week delay. Four days post-injury in these mice, coherent columns of LacZ-positive WAE cells emanated from adjacent crypts towards the wound center (Fig. 1C and fig. S2A) (6). At day 8 post-injury, distinctive LacZ-positive epithelial channels emanated from adjacent crypts towards the center of the wound (Fig. 1, C and D and fig. S2A). At later time points (14 and 28 days post-injury), clusters of LacZ-positive crypts were located within the original wound bed site (Fig. 1, C and E). Sections of these LacZ-positive crypts showed a return to homeostasis; differentiated epithelial cells (i.e. goblet cells) were present in the upper crypt similar to crypts in non-wounded areas (fig. S2B). These experiments showed that new colonic crypts can originate from crypts adjacent to the wound site and suggest that epithelial stem cells migrated through distinctive channels to enter the wound bed (fig. S2C).
At day 6 post-injury, we readily observed multiple invaginations of wound channels suggesting that multiple fission events occurred to produce new crypts (Fig. 1A). Comparison of transcript abundance (10) of budding wound channels (day 6 post-injury) to crypts in uninjured areas showed that the wound channel epithelium contained highly proliferative cells (fig. S3A) enriched in Wnt signaling transcripts (canonical and non-canonical combined) (fig. S3, A and B). As canonical Wnt signaling is critical for intestinal stem cell homeostasis (11-14) and non-canonical Wnt signaling plays a role in intestinal development (15), we screened expression of 19 Wnt ligands and 4 R-spondin co-activators by RT-PCR (fig. S3C). Interestingly, mRNAs encoding the non-canonical Wnt ligand Wnt5a were significantly enriched in the wound bed compared to the adjacent uninjured mucosa (Fig. 2, A and B; fig. S3, D and E).
Non-canonical Wnts expressed in undifferentiated mesenchymal cells affect tissue regeneration in hydra and zebrafish (16, 17). Similarly, in situ hybridization showed an expanded population of Wnt5a-positive stromal cells in the colonic wound bed (Fig. 2C and fig. S4) compared to uninjured mucosa (Fig. 2C) (18). Importantly, a sub-population of Wnt5a-positive cells localized near wound channels. Similar to other injury models, the Wnt5a-positive stromal cells in the colonic wound bed did not express markers of differentiation including α-smooth muscle actin, (myofibroblasts) or β-catenin (endothelial cells) (fig. S5, A and B). Interestingly, we noted an additional subpopulation of Wnt5a-positive cells beneath the mucosal wound bed in the serosal area (fig. S6A). During development, one potential source of undifferentiated stromal progenitor cells in the mouse intestine is mesothelial cells that form the serosa (the outer surface of the intestinal tube) (19). We performed a genetic lineage tracing experiment for mesothelial cells using WT1CreERT2: Rosa26R mice (20). Tamoxifen injection activates Cre recombinase in mesothelial cells that in turn marks these cells by LacZ. A population of Wnt5a-positive cells was derived from these WT1-marked cells at day 6 post-injury (fig. S6, B to E) suggesting Wnt5a-positive cells are in part derived from mesothelial cells.
We next examined the association of Wnt5a-positive cells with wound channel epithelial cells. During injury repair, wound channels were comprised of an expanded population of proliferative (Ki-67-positive), canonical Wnt-active (Axin2-positive; 21) epithelial cells. Strikingly, Wnt5a-positive cells localized to clefts at the base of wound channels, suggesting areas of nascent crypt formation (Fig. 2D). In addition, Wnt5a-positive cells also were located adjacent to non-proliferative wound channel epithelial cells (Fig. 2E). This was specific for wound channels, as in uninjured areas, Wnt5a-positive mesenchymal cells were not associated with crypt bases that contain canonical Wnt-active epithelial cells (Fig. 2D). These results suggested that Wnt5a-positive mesenchymal cells may induce new crypt formation by locally inhibiting proliferation of the stem/progenitor cell population within the wound channel.
To test this hypothesis in vivo, we generated a Wnt5a conditional knockout allele (fig. S7, A-D). We crossed Wnt5aflox mice with CAGGCreER™ (22) or Ubc-Cre-ERT2 (23) transgenic mice for global cellular targeting and generated CreERT-expressing Wnt5aflox/flox mice (Wnt5ako/ko) as well as CreERT-expressing Wnt5aflox/+ and Wnt5a+/+ mice (controls). After serial tamoxifen injections to activate Cre recombinase, we created colonic mucosal injuries and analyzed repair. Wnt5ako/komice generated by both methods contained normal appearing WAE cells (fig. S7E), but contained abnormal wound channels at day 6 and 8 post-injury. As compared to uninjured controls, at day 6 post-injury, Wnt5ako/ko wound channels contained significantly fewer invaginations (Fig. 2, F and G) and at day 8 post-injury, they did not develop into new crypt-like structures (Fig. 2H). These results showed that Wnt5a has a crucial role in the proper formation and eventual subdivision of wound channels into crypts (Fig. 2I).
To test the direct effects of Wnt5a on wound channel epithelium, we established an in vitro culture system that mimicked wound channels. Conditioned media from an L cell line expressing Wnt3a, R-spondin3 and noggin (L-WRN) (11, 13, 14) maintained highly proliferative, epithelial colonic stem/progenitor cells (Lgr5-positive; 24) as organoid spheres in culture (fig. S8, tables S2 and S3); these properties were similar to in vivo wound channel epithelial cells. Interestingly, Wnt5a-soaked beads (fig. S9) induced clefts within colonic organoids more frequently than control beads (Fig. 3, A and B). Epithelial proliferation in areas of contact with Wnt5a-soaked beads was suppressed compared to control beads (Fig. 3C). Furthermore, addition of Wnt5a to the organoid culture medium suppressed colonic sphere formation stimulated by Wnt3a and R-spondin1 (Fig. 3D). Wnt5a decreased Ki-67 and Lgr5 expression in a dose-dependent manner (fig. S10A). However, Axin2 expression was not suppressed by Wnt5a (fig. S10A), in contrast to previous findings that suggest Wnt5a antagonizes canonical Wnt signaling (25-28). The connection between non-canonical Wnt signaling and the cell cycle is unclear. Increased expression of cyclin-dependent kinase (Cdk) inhibitors can inhibit epithelial proliferation. We found that Wnt5a increased expression of multiple Cdk inhibitors, most notably p15INK4B (Cdkn2b) in treated colonic epithelial organoids (fig. S10B). These results show that a focal source of non-canonical Wnt initiates a critical intermediary step to reestablish homeostasis of the colonic epithelium.
Because p15INK4B expression can be induced by TGF-β (29), a classical inhibitor of epithelial proliferation (30), we tested if Wnt5a activated the TGF-β signaling pathway. In colonic organoids, recombinant TGF-β1 suppressed proliferation and induced Serpine1 (PAI-1) expression indicating Smad transactivation (31) downstream of the TGF-β type 1 receptor (fig. S11, A and B). Surprisingly, Wnt5a also stimulated Serpine1expression in a dose dependent manner (Fig. 4A) and enhanced Smad3 phosphorylation/nuclear localization (Fig. 4B). In injured Wnt5ako/ko as compared to control mice, the loss of Wnt5a expression was associated with diminished phosphorylation of Smad3 in the stem/progenitor population at the base of wound channels (Fig. 4, C and D and fig. S11, C and D). The action of Wnt5a required kinase activity of the TGF-β receptor as SB431542, a kinase inhibitor for TGF-β type 1 receptor, suppressed all Wnt5a effects on cell growth as well as Ki-67 and Serpine1 expression (Fig. 4E and fig S11E). Thus, Wnt5a expression is required to potentiate TGF-β signaling at the base of wound channels and mimicked the effects of Wnt5a in vitro.
Despite sharing several similar components, the canonical and non-canonical Wnt signaling pathways utilize distinct co-receptors, LRP5/6 and ROR1/2, respectively, (32). Intestinal organoids exclusively expressed Ror2 (fig. S12) and Ror2−/−and Wnt5a−/− mice similarly display defective gut elongation (15, 33). Using colonic organoids with shRNA knockdown of Ror2 (fig. S13, A-C), we found that Wnt5a and TGF-β1showed diminished Serpine1induction (Fig. 4F) indicating that the activation of the TGF-β signaling pathway by Wnt5a was mediated through Ror2. Interestingly, Ror2 knockdown did not affect growth inhibition of Wnt5a and TGF-β1 (fig. S14), suggesting that TGF-β inhibited the cell cycle in a transcription-independent manner as previously described (34) and that the signaling through Ror1/2 enhanced transcriptional activity of the Smad protein complex. Although the core components of the TGF-β signaling pathway consisting of type I and II receptors and Smad proteins are relatively simple, regulatory mechanisms, including protein modification and translocation, are highly complex (35). This experimental system will help identify more precise molecular mechanisms of cell cycle inhibition by non-canonical Wnt and TGF-β.
Here we show that wound channels are a critical intermediate structure during colonic epithelial wound repair. These channels undergo Wnt5a-mediated subdivision that utilizes a novel mechanism to potentiate TGF-β signaling. The effects of Wnt5a are primarily mediated by focal inhibition of proliferation, though we cannot rule out effects on cell polarity/asymmetric cell division. The process of crypt regeneration appears to re-utilize elements of a fetal developmental program (15, 36). Even though a colonic mesenchymal niche is not yet defined, our findings suggest that Wnt5a-positive cells are a critical part of this niche during injury repair as they affect the organization of the regenerating epithelium. We propose that such epithelial-mesenchymal interactions during repair will occur in other tissues and organisms (1) such as hair follicles that elevate specific Wnt family members during regeneration (37).
We thank R. Kopan for comments on the manuscript. We thank W. Pu for reagents. We thank R. Head and C. Storer for the microarray analysis. This work was funded by the NIH (DK90251), the Pew Scholars Program in the Biomedical Sciences, 5T35DK074375 (Trans-NIDDK Short-Term Training for Medical Students), the Washington University Digestive Disease Research Core (NIH P30-DK52574), and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Microarray data is available on Array Express (accession # E-MTAB-1175). MTAs will be required for the acquisition of the Wnt5af/f mice from the National Cancer Institute and the L-WRN cell line from the Washington University Medical School. The data presented in this manuscript are tabulated in the main paper and the supplementary materials.