BioProject

The gastrointestinal epithelial crypts are clonal units with a high cell turnover, driven by a small population of long-lived, Lgr5-expressing stem cells located in the crypt base. More...

The gastrointestinal epithelial crypts are clonal units with a high cell turnover, driven by a small population of long-lived, Lgr5-expressing stem cells located in the crypt base. Despite this, depletion of Lgr5+ cells does not lead to severe pathology. Instead, other cell populations, such as secretory and enterocyte precursors are able to de-differentiate, replace Lgr5+ cells, and regenerate the crypt. However, the signals that regulate this epithelial plasticity are not well understood. Here we illuminate the hierarchical organization and regulation of stem cell plasticity in the colonic crypt. Using in vivo lineage tracing, we find that the classic Wnt target gene Axin2 is expressed in colonic Lgr5+ cells and adjacent Lgr5-negative cells that give rise to entire crypts upon Lgr5+ cell depletion. The identity of Lgr5+ cells is controlled by by R-spondin 3 (Rspo3) produced by myofibroblasts and the recovery of Lgr5+ cells upon depletion required functional Rspo3 signaling. In contrast, expression of Axin2 and the reserve stem cell function of Axin2+ cells is not dependent on Rspo3. Accordingly, upon knockout of Rspo3, Lgr5-negative Axin2+ cells are able to compensate the loss of the Lgr5+ cell compartment and maintain epithelial integrity. In contrast, Rspo3 is essential for maintaining epithelial integrity in the context of injury of the entire Axin2+ cell pool, as observed in the context of DSS colitis. We demonstrate that DSS induces a progressive loss of the Lgr5+ stem cells as well as Axin2+ Lgr5-negative reserve stem cell compartment. While Rspo3 deficient animals are unable to withstand this injury, in Rspo3 competent animals, crypt homeostasis and regeneration are fueled by the remaining Axin2-negative, differentiated cells. These cells can be reprogrammed by Rspo3 to proliferate and act as stem cells. Thus, we identity Rspo3 as a major regulator of the Lgr5 stem cell signature. While loss of this signature can be compensated by Rspo3-independent reserve stem cells under healthy conditions, it is essential for re-establishment of epithelial integrity upon crypt injury. Overall design: Tamoxifen (Sigma) was injected intraperitoneally into mice either as single dose or on three consecutive days (4 mg/25 g bodyweight, diluted in 200 µl corn oil) at the indicated time points to deplete Rspo3 expression. C57BL/6 Axin2CreErt2/Rosa26-tdTomato cells were sorted using the BD FACSAria II. Cells were sorted for tdTomato, DAPI and ESA expression. Td Tomato was used as positive marker for Axin2 expressing cells. Microarray experiments were performed as dual-color hybridizations on Agilent whole mouse catalog 4x44K (Agilent-014868) arrays. To compensate for dye-specific effects, a dye-reversal color-swap was applied. Less...