O2 regulates stem cells through Wnt/β-catenin signalling

Abstract

Stem cells reside in specialized microenvironments or 'niches' that regulate their function. In vitro studies using hypoxic culture conditions (<5% O2) have revealed strong regulatory links between O2 availability and functions of stem and precursor cells. Although some stem cells are perivascular, others may occupy hypoxic niches and be regulated by O2 gradients. However, the underlying mechanisms remain unclear. Here, we show that hypoxia inducible factor-1α (HIF-1α), a principal mediator of hypoxic adaptations, modulates Wnt/β-catenin signalling in hypoxic embryonic stem (ES) cells by enhancing β-catenin activation and expression of the downstream effectors LEF-1 and TCF-1. This regulation extends to primary cells, including isolated neural stem cells (NSCs), and is not observed in differentiated cells. In vivo, Wnt/β-catenin activity is closely associated with low O2 regions in the subgranular zone of the hippocampus, a key NSC niche. Hif-1α deletion impairs hippocampal Wnt-dependent processes, including NSC proliferation, differentiation and neuronal maturation. This decline correlates with reduced Wnt/β-catenin signalling in the subgranular zone. O2 availability, therefore, may have a direct role in stem cell regulation through HIF-1α modulation of Wnt/β-catenin signalling.

Figure 1. Hypoxia activates Wnt/β-catenin signalling in mouse embryonic cells

a, ES and P19 EC cells transiently transfected with TOP-Flash and pRL-SV40 plasmids were grown either in normoxia (N, 21% O₂) or hypoxia (H, 1.5% O₂ for 16h) (n=9). b, β-catenin and LEF-1/TCF-1 analysis in ES cells under indicated O₂ conditions by western blot analysis of nuclear extracts. CREB served as a loading control, c, qRT-PCR analysis of Wnt target gene levels in hypoxic ES cells relative to 18S rRNA levels, normalized to normoxic cells (n=9). d, TOP-Flash activity in ES and P19 EC cells treated with 200 nM Broraoindirubin-3' oxime (B) and cultured either in hypoxia or normoxia (n=9). e, qRT-PCR analysis of Wnt-3a CM (W) treated ES cells show increased induction of Wnt target genes under hypoxia compared to normoxia (n=6). f, Severe reduction of reporter activity in ES and P19 EC cells transfected with pools of siRNA against β-catenin at indicated O₂ levels (UT: untransfected, tSi: target siRNA, cSi: control siRNA) (n=6) (left), and western blot analysis of silenced β-catenin 48 h post transfection (right), g, Growth rate of ES cells treated with Wnt-3a CM, or Wnt inhibitor DKK-1 (300 ng ml⁻¹) under indicated O₂ conditions (n=3). h, TOP-Flash activity in ES cells treated with DKK-1 (300 ng ml⁻¹) (n=3). *= P <0.05, **= P <0.005., Student's t-test. Error bars represent S.D..

Figure 2. HIF-1 (HIF-1α/ARNT complex) mediates hypoxia induced Wnt signalling in embryonic cells

a, Hif-1α^−/− ES cells (middle) show attenuated TOP-Flash activity under hypoxia as compared with hypoxic Hif-1α^+/+ cells (left). Increased reporter activity in normoxic ES cells treated with DFX (16 h) (right) (n=9). b, TOP-Flash activity in Hif-1α^+/+ and Hrf-1α^−/− cells stimulated with Wnt-3a CM (W) and cultured under indicated O₂ conditions (n=9). c, TOP-Flash activity is attenuated in hypoxic Arnt^−/− ES cells, and rescued in hypoxic Amt^Res cells (Arnf^−/− cells with restored Arnt expression) compared to hypoxic Arnt^+/+ cells (n=9). d, qRT-PCR analysis of Hif-1α^−/− cell extracts for Wnt target gene expression (n=6). e, Western blot analysis of β-catenin, LEF-1 and TCF-1 proteins in whole cell extracts of null cells (indicated by the absence of specific protein) or corresponding wild type cells cultured either under normoxia or hypoxia. Actin served as the loading control. f, Immunoprccipitation with LEF-1 antibody was performed on nuclear extracts of normoxic or hypoxic ES cells. CREB served as the loading control. g, ES cells were cultured under 21% or 1.5% O₂ for 16 h and then assayed by ChlP. Following IP with antibody against HlF-1α or isotype control, DNA extracts were assessed by qRT-PCR. Results of each genomic region tested (upper) are expressed as fold difference between HIF-1α IP and mouse IgG control (lower). Lef-1 coding region (I.C) served as a control (n=4). *= P <O.05, **= P <0.005., Student's t-test and one-way ANOVA (b). Error bars represent S.D..

Figure 3. O₂ regulation of Wnt/β-catenin signalling is differentiation stage specific

a, ES and P19 EC cells were treated with (+) or without (−) N2B27 neuronal growth and differentiation supplements, and monitored for the formation of neuronal progenitors (NP) through the expression of neuronal marker β-tub III. b, ES and P19 EC cell-derived NPs demonstrate attenuated TOP-Flash reporter activity under hypoxia compared to hypoxic undifferentiated controls (n=3) c, ES cell dcrived-NPs were cultured under normoxia or hypoxia for 16 h and assessed for Lef/Tcf gene expression by qRT-PCR. (n=3).**= P <0.005., Student's t-test. Error bars (b–c) represent S.D.. d, Western blot analysis of nuclear extracts of ES-Lef-1 and the corresponding control virus (VC) transduced ES cells cullured under normoxia. CREB served as the loading control. e, Schematic representation of a model wherein hypoxia (via H1F-1α) induces β-catenin transcriptional effectors, Lef-1 and Tcf-1, resulting in increased nuclear translocation of β-catenin (? indicates mechanism is unknown), subsequent interaction between β-catenin and LEF-1/TCF-1 and activation of Wnt target genes.

Figure 4. Deletion of Hif-1α in vivo suppresses adult hippocampal neurogenesis

a, Wnt activity in the adult dentate gyrus (DG) is marked by β-galactosidase (β-gal) immunostaining (red) in BAT-GAL reporter mice. Arrowheads indicate the subgranular zone (SGZ) in the DG. b, Hypoxic regions within the adult hippocampus marked by pimonidazole staining (HP, green) include the adult DG. c, The SGZ of the DG is marked by the stem cell marker Sox2. d, Colocalization of pimonidzole with Sox2⁺ cells indicates a hypoxic SGZ. Arrowheads (c–d) indicate the orientation of the SGZ of the granule cell layer (GCL). e, Laminin staining marks blood vessels in the DG, and in the adjacent hippocampal Oriens layer (Or) (f). g, Quantification of the nuclei: blood vessel ratio in the DG, Or and cerebcllar granule layer (CG) (n = 3; 5 sections per animal, **=P <O.005., Student's t test). Error bars represent S.E.M.. h–i, X-gal staining of the DG of Hif-1α^f/f, BAT-GAL^Tg (h) and gHif-1α^Δ/Δ, BAT-GAL^Tg (i) animals (n=3; 5 sections per animal). j, qRT-PCR analysis of hippocampal extracts for Wnt target genes (Lef-1, Axin2 and Dkk4) (n=3–5 per group). k, o, Significant reduction of DCX⁺ cells (arrows) in gHif-1α^Δ/Δ SGZ and GCL compared to control Hif-1α^f/f aniirials (n=7). l, p, DCX⁺ cells with neurtees are significantly fewer in gHif-1α^Δ/Δ DG as compared to gHif-1α^f/f DG. DCX⁺ cells with processes > 20 μM were counted positive for neurites (n=7). m, n, q, Lack of neuronal Hif-1α in the SGZ reduces NSC proliferation as indicated by BrdU⁺ cells (arrows) (m, q left panel), and NSC differentiation as indicated by DCX⁺ cells (arrows indicate DCX⁺ cells with neuritis, arrowheads indicate the direction of SGZ) (n, q right panel) (n=6). q, Quantification of BrdU⁺ and DCX⁺ cells in nHif-1α^Δ/Δ DG treated with SB216763 (2mg Kg⁻¹) every other day for 2 weeks. Vehicle treated nHif-1α^Δ/Δ and untreated nHif-1α^f/f mice served as controls (n=6). *= P <0.05, **= P <O.005., Student's t-tost (j, o, p) and one-way ANOVA (q). Error bars (j, o–q) represent S.E.M..

Figure 5. Neuronal Hif-1α regulates neurogenesis through Wnt/β-catenin signalling

a, E14 NSCs (E-NSC) or adult NSCs (A-NSC) isolated from 4–6 week old hippocampus were cultured as neurospheres, and employed for TOP-Flash assay under normoxia or hypoxia (n=6). b, Transfection efficiency for NSC TOP-Flash assays was monitored with GFP in 2-day post disassociation neurospheres (white arrows in phase). c, Neurospheres were assayed for progenitor markers including Nestin (a upper panel) and Sox2 (a lower panel). d, TOP-Flash assay in differentiated E14 embryonic (dE-NSC), or adult NSCs (dA-NSC) cultured under normoxia or hypoxia (n=6). e, qRT-PCR analysis of Lef-1 levels in A-NSCs or differentiated A-NSCs (dA-NSC) transfected with HIF-1α TM plasmid or corresponding vector control (VC) (n=3). f, Differentiation of neursopheres was assessed by the expression of differentiation markers including β-tublll (f upper panel) and DCX (f lower panel). *= P <0.05, **= P <0.005., Student's t-test (a, d–e). Error bars (a, d–e) represent S.D..