The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1 + ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1 + ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5 + ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.R-spondin | Dickkopf-1 | intestinal regeneration T he G protein-coupled receptor Lgr5 and the Polycomb group protein Bmi1 are two recently described molecular markers of self-renewing and multipotent adult stem cell populations residing in the crypt of the small intestine, capable of supporting regeneration of the intestinal epithelium (1, 2). Despite their similar ability to functionally repopulate the intestinal epithelium as demonstrated by independent in vivo lineage tracing experiments in reporter mice, the intestinal stem cells (ISCs) identified by these two molecular markers are spatially distinct. Whereas Lgr5 + ISCs are crypt base columnar (CBC) cells (1, 3) interspersed between Paneth cells and expressed throughout the intestine, Bmi1 + ISCs are mostly restricted to the "+4" cell position abutting the uppermost Paneth cell in proximal small intestine crypts (2). Lgr5 + ISCs are actively cycling (1), equipotent, and contribute to intestinal homeostasis by neutral drift competition (4-6). By comparison, Bmi1 + ISCs are less well characterized, and because of the lack of direct evidence, their cell cycle status is variably ascribed to be rapidly (7) vs. slowly cycling (8). It has been suggested that Bmi1 and Lgr5 mark an overlapping and possibly identical or redundant population of ISCs (5, 7, 9); however, no direct exploration of their functional similarities and differences has been performed. Further, it is unknown how Bmi1 + and Lgr5 + ISCs relate to a proposed model in which the intestine differentially uses an actively cycling ISC population during homeostasis and a distinct quiesce...

Summary How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage and stromal development from a population of highly pure, post-natal skeletal stem cells (mouse Skeletal Stem Cell, mSSC) to its downstream progenitors of bone, cartilage and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in non-skeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation towards bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ’s structure has been restored. The Wnt signaling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. The mechanisms that counterbalance Wnt-driven proliferation are poorly understood. We demonstrate here that YAP, a protein known for its powerful growth-inducing and oncogenic properties1-2, has an unexpected growth-suppressive function restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells (ISCs) and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signaling independently of the APC/Axin/GSK3β complex partly by limiting the activity of Dishevelled (DVL). DVL signals in the nucleus of ISCs and its forced expression leads to enhanced Wnt signaling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas (CRC) and its expression can restrict the growth of CRC xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing with no requirement for a quiescent stem cell sub-population. By means of double–labeling RNA in situ hybridization, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self renewal.

SUMMARY The canonical Wnt/β-catenin signaling pathway governs diverse developmental, homeostatic and pathologic processes. Palmitoylated Wnt ligands engage cell surface Frizzled (Fzd) receptors and Lrp5/6 co-receptors enabling β-catenin nuclear translocation and Tcf/Lef-dependent gene transactivation1–3. Mutations in Wnt downstream signaling components have revealed diverse functions presumptively attributed to Wnt ligands themselves, although direct attribution remains elusive, as complicated by redundancy between 19 mammalian Wnts and 10 Fzds1 and Wnt hydrophobicity2,3. For example, individual Wnt ligand mutations have not revealed homeostatic phenotypes in the intestinal epithelium4, an archetypal canonical Wnt pathway-dependent rapidly self-renewing tissue whose regeneration is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs)5–9. R-spondin ligands (Rspo1–4) engage distinct Lgr4-6 and Rnf43/Znrf3 receptor classes10–13, markedly potentiate canonical Wnt/β-catenin signaling and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo8,14–17. However, the interchangeability, functional cooperation and relative contributions of Wnt versus Rspo ligands to in vivo canonical Wnt signaling and ISC biology remain unknown. Here, we deconstructed functional roles of Wnt versus Rspo ligands in the intestinal crypt stem cell niche. We demonstrate that the default fate of Lgr5+ ISCs is lineage commitment, escape from which requires both Rspo and Wnt ligands. However, gain-of-function studies using Rspo versus a novel non-lipidated Wnt analog reveal qualitatively distinct, non-interchangeable roles for these ligands in ISCs. Wnts are insufficient to induce Lgr5+ ISC self-renewal, but rather confer a basal competency by maintaining Rspo receptor expression that enables Rspo to actively drive and specify the extent of stem cell expansion. This functionally non-equivalent yet cooperative interplay between Wnt and Rspo ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precision control of tissue regeneration.