Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance

Extracellular signals such as Notch, Wnt and Hedgehog have been linked with the self-renewal and maintenance of HSCs and progenitors^{10, 11}. Notch proteins are highly conserved cell surface receptors that regulate development¹² and are mutant in a variety of cancers, including leukemia¹³ and breast cancer^{14, 15}. Notch is a single-pass transmembrane receptor that is activated when its extracellular domain interacts with ligands of the Delta and Serrate families. Ligand-receptor interactions lead to cleavage and release of the intracellular fragment of Notch, which enters the nucleus and associates with the transcriptional repressor CBF-1 (also called CSL, for 'CBF-1 and RBP-J in mammals, Suppressor of Hairless in Drosophila and Lag-1 in C. elegans')^{12, 15, 16}. In association with the transcriptional coactivator mastermind-like 1 (MAML1)¹⁷, the intracellular fragment of Notch binds to and converts CBF-1 to a transcriptional activator, thereby initiating expression of target genes.

Evidence also suggests that Wnt signaling accomplishes an important regulatory function in hematopoietic progenitors and stem cells during fetal and adult development¹¹. Wnt proteins constitute a large family of secreted signaling molecules that are expressed in diverse tissues and influence multiple processes in vertebrate and invertebrate development²¹. In addition to the importance of the Wnt pathway in normal development, dysregulation of the Wnt pathway can have potent oncogenic effects in tissues such as colon, breast, prostate and skin^{11, 22, 23, 24}. Wnt proteins act by binding to the Frizzled family of seven-pass transmembrane proteins²⁵ as well as to proteins of the low-density lipoprotein receptor−related protein family, LRP5 and LRP6 (refs. 26, 27, 28). In the absence of a Wnt signal, -catenin is associated with a large multiprotein complex (the 'destruction complex'), which includes the scaffold protein Axin and the serine-threonine kinase glycogen synthase kinase 3 (GSK3). In this complex, -catenin is phosphorylated and is thereby targeted for ubiquitination and degradation²¹. Axin is a key negative regulator of the pathway as it facilitates GSK3-mediated phosphorylation and degradation of -catenin. Binding of Wnt proteins to their receptors inhibits phosphorylation of -catenin by GSK3, resulting in stabilization and accumulation of -catenin in the cytosol²⁹. -catenin then translocates to the nucleus, where it binds to members of the LEF-TCF family of transcription factors. LEF-TCF proteins are normally associated with the transcriptional repressor Groucho^{30, 31}. Binding of -catenin relieves this repression and allows LEF-TCF factors to induce expression of the appropriate target genes³².

Exposure of mouse and human hematopoietic progenitors to conditioned media containing Wnt proteins results in an increase in immature colony formation in vitro^{33, 34}. In addition, purified Wnt proteins and viruses with genes encoding activated -catenin enhance self-renewal of murine HSCs in vitro and HSC reconstitution in vivo^{35, 36}. The Wnt pathway is also required for HSC maintenance, as expression of the Wnt inhibitor Axin leads to inhibition of HSC proliferation and viability in vitro and reduced reconstitution in vivo^{35, 36}. The effects of Wnt signaling on HSCs in mice have been recapitulated in a nonobese diabetic−severe combined immunodeficient xenotransplant model, in which delivery of Wnt5A conditioned medium in vivo results in increased reconstitution by human HSCs³⁷.

Figure 1. Notch signaling is highly active in HSCs and reduced in differentiated cells. (a) Bone sections from TNR mice were stained with antibodies to c-Kit and GFP. Localization of c-Kit+ cells (red) undergoing active Notch signaling (GFP+; green) in the bone marrow cavity (left) and the trabecular bone region (right). Arrowheads point to GFP+c-Kit+ cells. Dashed line indicates the bone marrow−bone boundary. (b) HSC marker and GFP expression in bone marrow cells from wild-type or TNR mice. Numbers indicate percentage of cells in outlined areas. Data are from one representative wild-type mouse (left) and TNR mouse (right) (n > 20 for each). (c) Average frequency of GFP+ cells in KLS (n = 33) versus lineage-committed (n = 16) populations from TNR mice. The GFP background of wild-type cells was subtracted for each experiment before averaging. Data represent the average s.e.m. *, P < 0.0001. (d) Bone marrow cells of TNR and wild-type mice, loaded with Hoechst 33342. Left, side population (SP) region; right, side population cells with the highest dye efflux (SP-tip). Dotted lines, wild-type GFP expression; solid lines, TNR GFP expression. Percentages in dot plots indicate percent cells in outlined areas; numbers above bracketed lines indicate percentage of GFP+ cells. Results are representative of two independent experiments. (e) Cells from bone marrow, blood, spleen and thymus of TNR mice analyzed for GFP and lineage-specific markers. Data represent the average s.e.m. percentage of GFP+ cells (n = 5 for bone marrow and blood, n = 4 for spleen and thymus, and n = 3 for double-negative (DN) subpopulations). Background GFP expression was subtracted for each value. Full Figure and legend (61K)

Figure 2. Notch signaling is downregulated during differentiation and defines a more primitive subset within the HSC population. (a) Left, expression of lineage markers and GFP of KLS GFP+ cells cultured in the presence of SLF and IL-3 for 3 d. Percentages indicate the frequency of GFP+ cells for Linneg/lo and Lin+ populations. Right, mean GFP fluorescence intensity of GFP+Linneg/lo or GFP+Lin+ populations. Results are representative of three experiments. (b) KLS GFP+ and KLS GFP- cells were sorted at a density of one cell per well into 96-well plates and were cultured in methylcellulose for analysis of in vitro colony-forming ability. Colonies were assigned scores for the total number of colonies (left) and the presence of single or multiple lineages (right). Data represent average s.e.m. of eight independent experiments. (c) 'CFU spleen' capacity of KLS GFP+ and KLS GFP- cells assessed as a ratio of day-12 colonies to day-8 colonies (CFU-S12/CFU-S8) after transfer into lethally irradiated syngeneic recipients. Data are representative of five independent experiments using 5−20 recipients per condition. Full Figure and legend (18K)

That Notch signaling marked the most primitive, multilineage cells suggested that the pathway may be important for maintenance of the undifferentiated state. If this hypothesis were correct, we reasoned that inhibition of the Notch pathway should lead to accelerated differentiation. We thus inhibited Notch signaling in HSCs by engineering retroviruses that express a dominant negative mutant form of xenopus suppressor of hairless (dnXSu(H); the xenopus homolog of CBF-1)⁴¹. This mutant protein has been shown to be a 'pan' (CBF-1-dependent) Notch signaling inhibitor in both mammalian and nonmammalian cells⁴². Additionally, given the early embryonic death (by day 10.5) of CBF-1-deficient mice⁴³, dnXSu(H) is an effective tool for abrogating Notch signaling. We confirmed expression of dnXSu(H) in HSCs by RT-PCR analysis after infection and found it decreased expression of the Notch target gene Hes1 (hairy and enhancer of split 1; Supplementary Fig. 6 online). HSCs in which CBF-1-dependent Notch signaling was inhibited showed accelerated differentiation compared with that of control cells (39% versus 20% Lin⁺; Fig. 3a and Supplementary Fig. 7 online). The cells generated in vitro after inhibition of Notch signaling were predominantly of the myeloid lineages, although we also noted some increase in B220⁺ cells (Fig. 3b). To determine whether other commonly used inhibitors of Notch signaling recapitulate the effects of dnXSu(H), we inhibited Notch signaling with a dominant negative form of MAML1 (dn(MAML1)), which has been shown to inhibit Notch signaling in several contexts¹⁷. After retroviral transduction, expression of dn(MAML1) in HSCs led to enhanced differentiation of HSCs (41% versus 24% Lin⁺; Fig. 3c). In addition to using dnXSu(H) and dn(MAML1), we treated KTLS cells with -secretase inhibitor II. This inhibitor blocks Notch signaling by preventing cleavage of the intracellular fragment of Notch from the full Notch receptor, a crucial step in the Notch signaling cascade^{10, 39, 44}. A higher fraction of HSCs incubated with -secretase inhibitor expressed lineage markers than did cells treated with vehicle control (34% versus 15% Lin⁺; Fig. 3d). The similar effects of -secretase inhibitor II, dnXSu(H) and dn(MAML1) suggest that the accelerated differentiation observed was due to inhibition of Notch signaling and that it was unlikely to be due to nonspecific effects of any one inhibitor.

Figure 3. Inhibition of Notch signaling in HSCs leads to accelerated differentiation. Freshly purified wild-type KTLS cells were infected with vector control retroviruses (Control) or with retroviruses producing dnXSu(H) (a,b) or dominant negative MAML1 (dnMAML; c). After 48 h of infection, cells were analyzed by flow cytometry for expression of GFP and lineage markers. Percentages indicate the frequency of GFP+Lin+ (top box) and GFP+Lin- (bottom box) cells. Results are representative of three to ten independent experiments. (b) Expression of the lineage markers B220, Mac-1 and Gr-1 on dnXSu(H)- or vector control−infected cells. Data represent average s.e.m. (n = 3). *, P < 0.02. (d) Purified wild-type KTLS cells were cultured in the presence of 5.0 M -secretase inhibitor II or vehicle control along with SLF (10 ng/ml). After 72 h in culture, cells were analyzed for lineage marker expression. Percentages indicate the frequency of Lin+ (top box) and Lin- (bottom box) cells. Results are representative of three independent experiments. Full Figure and legend (34K)

Figure 4. Inhibition of Notch signaling in vivo leads to depletion of the HSC population after long-term reconstitution. (a) Reconstitution of mice transplanted with control- or dnXSu(H)-infected HSCs (representative data). Percentages indicate the frequency of donor-derived cells (CD45.2+) for each of the outlined populations. (b) Average donor-derived KLS cells in mice transplanted with dnXSu(H)-infected cells or control transplants. *, P = 0.01; **, P = 0.05. Data represent the absolute number of CD45.2+ KLS cells s.e.m. in both transplant experiments; values were calculated by multiplying the observed KLS frequency by the estimated total number of bone marrow cells in host mice. Transplant 1 and 2 refer the first and second experiments, respectively, as described in Methods. Full Figure and legend (33K)

Table 1. Reconstitution efficiency of dnXSu(H)- or control-transduced HSCs Full Table

The data presented above suggest that Notch signaling is required for the prevention of lineage commitment and differentiation. As multiple signals are likely to be received by HSCs in context of the bone marrow niche, we were interested in determining whether Notch signaling has a dominant function relative to that of other signals in the maintenance of the undifferentiated state. We tested this specifically in context of Wnt signaling, which acts in synergy with SLF to promote HSC proliferation without substantial differentiation³⁵. First, we determined whether both Wnt and Notch signal in the same cells within the native microenvironment. Thus, we crossed TNR mice with Wnt reporter mice (Tcf optimal promoter -galactosidase (TOPGAL) mice)⁴⁵. TOPGAL transgenic mice, which have a minimal LEF-TCF promoter linked to the -galactosidase gene, express -galactosidase in response to Wnt signals. The trabecular bone area of the TOPGAL-TNR double-transgenic mice had a high frequency of cells that were double-positive for both -galactosidase and GFP expression (85% of TOPGAL⁺ cells and 65% of TNR⁺ cells were double positive; Fig. 5a). This result indicated that both Wnt and Notch signals were active in the majority of cells in the HSC niche and that a large fraction of cells used both pathways simultaneously. In addition, stimulation of HSCs with Wnt3A upregulated expression of Hes1 and Dtx1, downstream target genes of Notch signaling in HSCs (Fig. 5b), as well as Notch reporter activity in HSCs from TNR mice (data not shown). In comparison, of six Notch target genes tested, overexpression of the intracellular fragment of Notch itself upregulated only three target genes in HSCs: Hes1, Dtx1 and Hey1 (data not shown). These data suggest that Wnt contributes to the differential expression of known Notch targets in HSCs.

Figure 5. Inhibition of Notch signaling disrupts ability of Wnt signaling to maintain HSCs in an undifferentiated state. (a) Bone sections from TOPGAL-TNR double-transgenic mice stained with antibodies to -galactosidase (red) and GFP (green). In double-positive cells (white arrows), Wnt and Notch pathways are simultaneously activated. (b) Wild-type KLS cells were stimulated for 12 h with either soluble Wnt3A or vehicle control in the absence of any other growth factors, and G6pdx (G6PD), Hes1 (Hes-1) and Dtx1 (Deltex-1) expression was analyzed by real-time PCR. Data represent relative gene expression s.e.m. averaged from three to four independent experiments. *, P = 0.0002; **, P = 0.02. (c,d) KTLS cells transduced with dnXSu(H) or control retrovirus were re-sorted after 48 h based on expression of GFP (GFP+) and absence of lineage markers (Lin-) and were cultured for 3 d in the presence of SLF and Wnt3A. They were then analyzed by flow cytometry for (c) 5-bromo-2'-deoxyuridine (BrdU) incorporation and (d) propidium iodide (PI) staining and Annexin V binding. Results are representative of three independent experiments. (e) c-Kit+Linneg/lo (KL) cells transduced with control, dnXSu(H) or Axin retrovirus were analyzed for propidium iodide incorporation after 48 h of infection (n = 3). The percentage of propidium iodide−positive cells s.e.m. was significantly higher in Axin-infected cells than in control cells (*, P = 0.005). (f,g) KTLS cells were transduced, re-sorted and cultured as described for c,d. The extent of differentiation was measured by (f) lineage marker expression over 5 d and (g) expression of B lineage (B220) and myeloid lineage (Mac-1 and Gr-1) markers after 3 d of culture. Full Figure and legend (41K)

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Our observation that Notch signaling is active in HSCs and is important during the earliest stages of hematopoietic development is consistent with the results of other studies. For example, gain-of-function studies have shown that overexpression of the intracellular domain of Notch enhances HSC self-renewal^{18, 19}. Conversely, loss of Notch signaling impairs HSC development and function both in vitro and in vivo: -secretase inhibitor II reduces HSC frequency in the context of stromal cells³⁹ and Notch1 deficiency impairs the establishment and maintenance of hematopoietic stem cells^{49, 50}. Our finding that a critical function of Notch is to retard differentiation provides a mechanistic basis for how modulation of Notch signaling may control HSC numbers in these experiments. It also suggests that the function of Notch in the hematopoietic system is similar to its function in other systems^{51, 52}. The studies reported above as well as our results contrast with studies using interferon--inducible Notch1-deficient mice, which have shown that inducible loss of Notch fails to influence hematopoiesis⁵³. The reasons for the differences between these systems are unclear, but it is possible that interferon--induced deletion sets up a substantially different context for the study of HSC development. Another way to address the importance of Notch signaling in HSCs would be to analyze CBF-1-deficient mice; unfortunately, those mice die by embryonic day 10.5, so analysis of the effect of CBF-1 loss on HSCs has not been possible⁴³.

Our findings also indicate that the ability of Notch signaling to inhibit differentiation is dominant relative to Wnt and SLF proteins. Thus, whereas the proliferation and survival of HSCs exposed to Wnt and SLF proteins seem unaffected when Notch signaling is impaired, their ability to remain undifferentiated is substantially altered. Our findings do not preclude the possibility that a stronger Wnt signal, such as activated -catenin, may be able to overcome the consequences of loss of Notch signaling. However, it is likely that soluble Wnt better recapitulates physiological levels of Wnt signaling. The proliferative aspects of Wnt induced self-renewal may be driven by such genes as Ccnd1, Ccnd2 and Myc, which have been shown to be expressed in response to Wnt3A in HSCs (A.W.D. and T.R., data not shown) and in other systems^{54, 55, 56}.

One possible interpretation of the finding that Notch signaling is required for the influence of Wnt on HSCs is that Wnt signaling exerts its influence by activating the Notch pathway. Our observation that Wnt3A upregulates Notch target genes is consistent with this possibility. The idea of a hierarchical relationship between Wnt and Notch signaling is also supported by studies in drosophila showing that mutations in Notch can produce phenotypes that mimic Wg (wingless) mutant phenotypes⁵⁷ and by studies in cell lines showing that Wnt1 can upregulate Hes1 promoter activity⁵⁸. In both cases, the effects have been suggested to be mediated by GSK3, which can promote degradation of the intracellular fragment of Notch⁵⁸. Thus, Wnt signaling, by repressing GSK3, may lead not only to the accumulation of -catenin and activation of Wnt target genes but also to the accumulation of the intracellular fragment of Notch and activation of Notch targets such as Hes1. It is also possible that Wnt and Notch represent parallel pathways in HSCs, with Wnt enhancing proliferation and survival and Notch preventing differentiation. If this is the case, the observed upregulation of Hes1 and Dtx1 in HSCs may reflect the possibility that Wnt and SLF selectively promote survival or growth of HSCs that signal in response to Notch.

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HSCs were sorted from mouse bone marrow as described⁶⁸. For analysis of lineage-positive cells from bone marrow, spleen and thymus, single-cell suspensions were prepared from the organs. Blood cells were obtained from 0.5 ml of blood collected by cardiac puncture and diluted in 0.5 ml of 10 mM EDTA in PBS. Then, 1 ml of 2% dextran was added to each sample and samples were depleted of red blood cells by sedimentation for 45 min at 37 °C. Red blood cells were lysed with 1 RBC Lysis Buffer (eBioscience) before staining.

HSCs were sorted and re-analyzed based on surface marker expression of c-Kit and Sca-1, low expression of Thy-1.1, and Lin^lo to Lin^- expression. The combination of the following antibodies defined the lineage markers: 145-2C11 (antibody to CD3 (anti-CD3)), 53-7.3 (anti-CD5), GK1.5 (anti-CD4), 53-6.7 (anti-CD8), RB6-8C5 Ly-6G (anti-Gr-1), M1/70 (anti-CD11b, Mac-1), Ter119 (anti-erythrocyte specific antigen) and 6B2 (anti-B220). Other antibodies used included clones 2B8 (anti-CD117, anti-c-Kit), D7 (anti-Ly-6A/E, anti-Sca-1) and HIS51 (anti-CD90.1, anti-Thy-1.1). All antibodies were purchased from Pharmingen or eBioscience. Analysis and cell sorting were done on a FACSVantage (Becton Dickinson) at the Duke University Comprehensive Cancer Center Flow Cytometry Shared Resource.

dnXSu(H) and dominant negative MAML1 were cloned into the MSCV-IRES-GFP retrovirus expression vector. MSCV-IRES-GFP alone was used as control vector. Virus was produced by triple transfection of 293T cells with mouse stem cell virus constructs along with gag-pol and vesicular stomatitis virus G glycoprotein constructs. Viral supernatant was collected for 2 d and was concentrated 100-fold by ultracentrifugation at 50,000g. For viral infection, 25,000−50,000 HSCs were cultured overnight in a 96-well U-bottomed plate in the presence of X-Vivo15 medium (Bio-Whittaker), 50 M 2-mercaptoethanol, 2% FBS, 30 ng/ml of SLF and 30 ng/ml of Flt3 ligand. After 12−18 h, concentrated retroviral supernatant was added to the cells at a ratio of 1:5. Cells were then incubated at 32 °C for 12 h and were subsequently incubated at 37 °C for 36 h. Infected cells were then sorted based on their GFP expression for in vitro and in vivo assays. All cytokines were purchased from R&D systems.

For in vitro differentiation, 2,000 KLS GFP⁺ sorted cells were cultured in 96-well U-bottomed plates in medium containing X-Vivo15, 50 M 2-mercaptoethanol, 10% FBS, 10 ng/ml of SLF and 300 ng/ml of IL-3. After a 3-day culture period, the cells were stained for lineage markers and were analyzed by flow cytometry for lineage and GFP expression.

For assessment of the colony-forming capacity of Notch signaling cells, KLS GFP⁺ cells and KLS GFP^- cells were sorted at a density of 1 cell per well in 96-well plates and were cultured in complete methylcellulose medium (Methocult GF M3434 from StemCell Technologies). Colonies were assigned scores after 7 d of culture and were identified based on apparent morphological criteria. HSCs that were either retrovirus infected or were treated with -secretase inhibitor II (Calbiochem) were cultured in 96-well U-bottomed plates in medium containing X-Vivo15, 50 M 2-mercaptoethanol, 10% FBS, 10 ng/ml of SLF and Wnt3A (purified as described³⁶; estimated working concentration, 100 ng/ml). Proliferation was monitored by culture of HSCs for 48 h, pulsing with 10 M 5-bromo-2'-deoxyuridine (Sigma) for 12−18 h and staining of cells with anti-5-bromo-2'-deoxyuridine (BD Pharmingen). Cell death was determined by incubation with propidium iodide and Annexin V (BD Pharmingen) after 3 d in culture. The degree of differentiation of cultured cells was monitored at defined intervals by staining of HSCs for expression of lineage cell surface markers. All samples were analyzed by flow cytometry.

Freshly obtained bone specimens from TNR, TOPGAL-TNR and wild-type mice were decalcified, infiltrated with sucrose and embedded in optimum cutting temperature medium. Frozen sections were fixed in acetone and stored at -80 °C until staining. For immunostaining, sections were first blocked with 20% normal goat serum. For TNR bone analysis, samples were then incubated with c-Kit-specific biotinylated antibody (CalTag) and anti-GFP−fluorescein isothiocyanate conjugate (Molecular Probes), followed by incubation with streptavidin−Alexa 350 (Molecular Probes). For TOPGAL-TNR bone analysis, samples were then incubated with anti-GFP−fluorescein isothiocyanate conjugate as well as anti--galactosidase, followed by 7-amino-4-methylcoumarin-3-acetic acid secondary antibody. Samples were mounted using fluorescent mounting media and were viewed with appropriate filters.

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Competing interests statement:  The authors declare that they have no competing financial interests.