dna staining br Results br Discussion The

Results
Discussion The Wnt signaling pathway has emerged as the dominant self-renewal pathway for various adult-type stem cells and is required for maintenance of embryonic as well as induced pluripotent stem cells. In the hematopoietic system, only mild increased Wnt dosages result in higher stem cell activity; indeed the overall Wnt signaling levels in HSC are much lower than those found in intestinal, skin, or mammary gland stem cells. Nevertheless, complete loss of Wnt signaling leads to defective self-renewal as shown in secondary transplantations. This had led to interest in the use of Wnt signaling or factors that modulate Wnt signaling, such as prostaglandin E2 (PGE2) (Goessling et al., 2009) or GSK-3β inhibitors (Huang et al., 2012), for expansion of HSCs ex vivo. We previously demonstrated that Wnt signaling functions in a strictly controlled dosage-dependent fashion (Luis et al., 2011). As also shown by several other laboratories (Kirstetter et al., 2006; Ming et al., 2012) (Scheller et al., 2006), high Wnt levels in HSCs eventually lead to stem cell exhaustion and lack of reconstitution of irradiated recipients. In the current study, we used gene expression profiling to understand why Apc mutations that lead to high Wnt signaling (among other defects) in HSCs would lead to loss of repopulating capacity. Our results show, both at the genetic level and in functional assays, increased differentiation, diminished proliferation, and no effects on apoptosis. The much stronger differentiation toward mature blood linages coupled with loss of HSC proliferation (see also Figure S4) is expected to lead to lower reconstitution by HSCs. Collectively, these data explain the lack of maintaining bona fide stemness in Apc exon 15 deleted HSCs. Thus, instead of increased apoptosis of HSCs, here we offer another explanation for the loss of reconstitution capacity induced by high Wnt levels. An alternative interpretation of our data is that the observed consequences of Apc mutant dna staining are not Wnt but rather APC dependent. Apc encodes for a multifunctional protein involved in a broad spectrum of cellular functions (Gaspar and Fodde, 2004). To date, most Apc mutant mouse models are characterized by tumor phenotypes that depend completely on Wnt dosage. Apc1638T, the only targeted Apc mutation that does not affect Wnt signaling at all, results in homozygous viable and tumor-free animals, notwithstanding the deletion of the C-terminal third of the protein containing many functional domains (Smits et al., 1999, 2000). Deletion of only a few amino acids encompassing crucial Axin-binding motifs results in Wnt signaling activation, tumor formation, and lack of reconstitution by HSCs, as we have shown before (Luis et al., 2011). Finally, mutations affecting other members of the Wnt pathway, such as Gsk3β and β-catenin, result in levels of signaling activation and hematopoietic defects that are fully in agreement with our results (Goessling et al., 2009; Huang et al., 2009, 2012; Lane et al., 2010). Therefore, the most likely explanation is that specific levels of Wnt signaling are the major determinant of the observed differential effects on hematopoiesis. In addition, recent studies using recombinant Wnt3a also showed a dose-dependent effect on HSC biology (Famili et al., 2015) where high Wnt3a leads to loss of human HSC proliferation in vitro (Duinhouwer et al., 2015), underscoring the differential effects we also have observed with the different Apc alleles and correlating exactly with the Wnt dosages caused by these mutations. The finding that the Apc 15lox mutant leading to high Wnt signaling levels is associated with increased numbers of differentiated cells is not unprecedented. In the intestine, Wnt signaling induces maturation of Paneth cells that contain active β-catenin and Tcf4 (van Es et al., 2005), confirming that high Wnt signaling levels can drive differentiation processes. Other investigators have used a different system to increase Wnt signals in HSCs, namely overexpression of an oncogenic, constitutively active form of β-catenin (Ming et al., 2012). They showed an increase in apoptosis using annexin V/propidium iodide staining from 10% in WT LSK cells to 35% in high Wnt LSK cells. The reasons for the differences with our results could be due to differences in the systems used, although both are expected to lead to high Wnt signaling levels. Possibly activated β-catenin also negatively affects cell adhesion and homing properties thereby decreasing exposure to important survival signals leading to increased apoptosis. It is also noteworthy that enhanced survival signals are needed to have HSCs survive in the oncogenic β-catenin system. In addition, Li et al. (2013) have shown that Apc regulates the function of HSCs largely through β-catenin-dependent mechanisms, thus demonstrating that, in both systems, canonical Wnt signaling is the major factor.