br Experimental Procedures br Author Contributions All

Experimental Procedures
Author Contributions All authors fulfill the ICMJE guidelines for authorship. E.B.B., Y.L.T., Y.P.P., S.L., H.S.C., Q.D., and S.-B.J. performed the biological experiments. Y.L.T. and D.R. performed the bioinformatics part of the study. A.L., X.L., C.S., S.-B.J., and U.L. designed the study. S.-B.J. and U.L. conceived the study and U.L. was the lead writer of the manuscript. All authors critically read the manuscript, approved the final version, and agree to be accountable for all aspects of the work. Shared first authors: E.B.B. (Figures 2 and S2), Y.L.T. (Figures 4 and S4), Y.P.P. (Figures 1D and 1E).
Acknowledgments We wish to thank Agneta Birgitta Andersson and the BRECT core facility for excellent assistance with the xenograft experiments. This work was financially supported by the Swedish Cancer Society, the Swedish Research Council (DBRM and Project Grant), Knut och Alice Wallenbergs Stiftelse, Karolinska Institutet (BRECT), the Swedish Cultural Foundation of Finland, and K. Albin Johansson Foundation.
Introduction In many tissues, including the blood, intestine and skin, old knowing it are eliminated and replenished by newly developed cells from a small pool of stem cells. This rare population of stem cells is located in a specific microenvironment, the niche, and gives rise to several different lineages of abundant daughter cells (Mendez-Ferrer et al., 2010). The signals controlling the various stem cell fates (self-renewal, differentiation, quiescence, apoptosis, and others) are beginning to be elucidated. A number of evolutionary conserved pathways are important for the development and maintenance of adult stem cells, including Notch, bone morphogenic protein, hedgehog, fibroblast growth factor, transforming growth factor β, and Wnt signals (Blank et al., 2008). Among these pathways, the Wnt pathway is seen as a dominant factor in self-renewal of many types of adult stem cells (Reya and Clevers, 2005). Compared with the convincing studies on the role of Wnt signaling in adult stem cells in skin and gut, a role for Wnt in adult hematopoietic stem cells (HSCs) has proved much more difficult to demonstrate (reviewed in Luis et al., 2012). In studies reporting an important role for Wnt signaling in blood cells, Wnt seemed to be required for normal HSC self-renewal and therefore for efficient reconstitution after transplantation (Luis et al., 2011). Several types of Wnt signaling can be discerned often referred to as the canonical or Wnt/β-catenin pathway and the non-canonical pathways (reviewed extensively in Staal et al., 2008). In the absence of Wnt ligands, cytoplasmic levels of β-catenin are kept very low through the action of a protein complex (the so-called destruction complex) that actively targets β-catenin for degradation. This complex is composed of two negative regulatory kinases, including glycogen synthase kinase 3β (GSK-3β) and at least two anchor proteins that also function as tumor suppressor proteins, namely Axin1 or Axin2 and APC (adenomatous polyposis coli). APC and Axin function as negative regulators of the pathway by sequestering β-catenin in the cytoplasm. Hence, inactivating mutations in Apc lead to higher β-catenin protein accumulation among other important events controlled by APC. Activation of the pathway by Wnt leads to inactivation of the destruction complex allowing buildup of β-catenin and its migration to the nucleus. In the nucleus, β-catenin binds to members of the TCF/LEF transcription factor family, thereby converting them from transcriptional repressors into transcriptional activators. Initial attempts to overexpress a constitutively active form of β-catenin in HSCs led to an increase in proliferation and repopulation capacity upon transplantation into lethally irradiated mice (Reya et al., 2003). However, later studies using conditional overexpression of a stabilized form of β-catenin led to a block in multilineage differentiation, and the exhaustion of long-term HSCs (Kirstetter et al., 2006; Scheller et al., 2006). This resulted in anemic mice and eventually led to lethality, i.e., the opposite effect when compared with the improved transplantation setting reported earlier. These studies have created confusion concerning the importance of Wnt in maintaining numbers and integrity of HSCs. Similarly, not all loss-of-function studies have produced clear phenotypes. The Mx-Cre system has been used to drive deletion of β-catenin (Zhao et al., 2007) or both β-catenin and its homolog γ-catenin (Koch et al., 2008; Jeannet et al., 2008). However, no defects were reported in HSC function or cells within lymphoid tissues. Surprisingly, in vivo reporter assays revealed that the canonical Wnt signaling pathway was still active in HSCs despite the absence of both β- and γ-catenin (Jeannet et al., 2008). This could imply the existence of an alternative factor or generation of a hypomorphic allele permitting low levels of Wnt signaling that would negate hematopoietic defects. Heroic efforts to knock out the Porcn gene during hematopoiesis, which encodes an acyltransferase (porcupine) necessary for acylation of Wnts, enabling their secretion and binding to the frizzled receptors, have not resulted in hematopoietic defects; however, there also were no changes in Wnt signaling (Kabiri et al., 2015). The reasons for this are presently unknown, but incomplete deletion or the lack of need for Wnt secretion have been suggested (Oostendorp, 2015). This demonstrates the high complexity and difficulty in generating bona fide null mutants for canonical Wnts in the hematopoietic system. Together with studies in which Wnt activity in HSCs was reported to be close to zero (Fleming et al., 2008; Luis et al., 2009; Zhao et al., 2007), these findings suggest that complete absence of Wnt signaling is detrimental to HSC function, but that up to a quarter of normal activity is sufficient for normal function. Our recent findings suggest that these very different results in both gain-of-function and loss-of-function studies can be largely explained by differences in levels of Wnt signaling achieved in different experimental circumstances. That is, when Wnt signaling is slightly enhanced over normal levels, HSCs show improved reconstitution capacity. However, when HSCs express high levels of Wnt signaling, they completely fail to reconstitute irradiated recipient mice (Luis et al., 2011). Thus, different levels of activation of the pathway can account for the discrepancies in previous studies (Malhotra and Kincade, 2009).