SEARCH

SEARCH BY CITATION

Keywords:

  • Hematopoietic stem cells;
  • Progenitor cells;
  • Conditional knockout mouse;
  • Ets;
  • Tie2 and ER71

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The Ets transcription factor Er71 is an important regulator of endothelial and hematopoietic development during mammalian embryogenesis. However, the role of Er71 in adult hematopoiesis has remained unknown. We now first show that conditional deletion of Er71 in the hematopoietic system of adult mice results in a marked reduction (55%) in the number of hematopoietic stem cells (HSCs) that is likely due to increased cell death. Bone marrow transplantation (BMT) experiments further confirmed that Er71 is required for repopulation of HSCs. In addition, Er71+/− mice exhibited a slight decrease (37%) in the number of HSCs than those of Er71+/+ mice, indicating that the function of Er71 in HSC maintenance is dependent on gene dosage. Moreover, Er71 was shown to be required for Tie2 expression, which contributes to HSC maintenance. Our results thus suggest the role of a single transcription factor in controlling HSCs through regulation of Tie2 expression in adult animals. STEM CELLS 2011;29:539–548


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Hematopoietic stem cells (HSCs) are the only blood-forming cells capable of long-term self-renewal or differentiation into diverse lineages in vertebrate hematopoiesis. This process is characterized by a hierarchical cellular progression, in which cells with high plasticity reconstitute the more restricted states of the hematopoietic system [1]. Only few transcription factors have been identified as regulators of HSC activity during adult hematopoietic development [1–4]. The transcriptional regulatory network of HSC self-renewal thus remains largely undefined.

The Ets family of transcription factors has been found to contribute to hematopoiesis [5, 6]. The Ets transcription factor PU.1, for example, regulates myelo- and lymphopoiesis [7–9] and HSC self-renewal [10–12]. Tel (also known as Etv6), another Ets transcription factor, is a selective regulator of adult HSC survival [13], whereas Ets1 functions in lymphoid development [14, 15] and Fli1 has an essential role in megakaryopoiesis [16, 17]. The Ets transcription factor Er71, which was originally thought to be testis-specific, has also been implicated in the development of endothelial and blood lineages in mice [18–20]. Similarly, Etsrp, the ortholog of Er71 [19, 21, 22], plays a key role not only in vasculogenesis in zebrafish but also in the induction of myelopoiesis in zebrafish and Xenopus embryos [19, 21, 23, 24]. We previously showed that ablation of the Er71 gene in mice resulted in the death of embryos early in gestation; the embryos essentially lacked blood and vascular structures, and hematopoietic cells were undetectable [18]. Er71 has also been shown to be transiently expressed in the endocardium and endothelium of the developing mouse embryo, in which it specifies an endothelial or endocardial fate [20]. Er71 functions as a potent activator of several early endothelial genes, including Flk1, Tal1, Mef2c, Pecam1, and Tie2, by directly binding to their promoters, and it is a downstream target of signaling by bone morphogenetic protein, Notch, Wnt, and Nkx2-5 in the developing embryo [18, 20, 23].

Taken together, mouse and zebrafish studies highlight the developmental role of Er71. However, the role of Er71 in adult hematopoiesis has remained largely unexplored. To investigate the physiological role of Er71 in adult hematopoiesis further, we generated conditional mutant mice in which Er71 is deleted specifically in the hematopoietic system. We now show that Er71 contributes to normal HSC function and maintenance in a dose-dependent manner in adult mice. Furthermore, we found that Tie2 expression was markedly downregulated in Er71-deficient HSCs and that Er71 increases the activity of the Tie2 promoter. Together, our results indicate that Er71 regulates Tie2 transcription and thereby contributes to HSC maintenance in adults.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Ethics Statement

Animal care and experimentation were performed in accordance with procedures approved by the KAIST (Korea Advanced Institute of Science and Technology) Animal Care and Use Committee (KACUC).

Generation of Er71 Conditional Knockout Mice

To construct the conditional targeting vector, we subcloned the PGK-neo gene into the EcoRI-XbaI site of the Er71-PGK-puro vector. The resulting vector contains loxP sites, the PGK-neo gene flanked by recognition sites for FLP recombinase, and the diphtheria toxin A gene for negative selection, and it was designed to remove exons 5 and 6 of mouse Er71. The targeting vector was linearized with NotI and introduced into AK7 ESCs by electroporation, and the cells were then subjected to selection in the presence of G418. G418-resistant clones were isolated after 10 days of culture, and homologous recombination was confirmed by Southern blot analysis of genomic DNA with 5′ and 3′ external probes. We identified six correctly targeted clones, which were designated Er71+/fl-neo because they contain an Er71 allele with loxP sites and the neo gene. ESCs of three independent clones containing the targeted allele were then injected into blastocyst-stage embryos, the resulting chimeric male mice were crossed with C57BL/6 females, and germline transmission of the targeted allele was confirmed. After germline transmission, neo gene was removed by crossing with Flp deleter mice. The resulting Er71fl/+ mice were born in the expected numbers and appeared to develop normally. Moreover, Er71fl/fl mice were viable and phenotypically normal, consistent with the floxed Er71 allele being functionally wild type (WT). For conditional deletion of Er71 in vivo, Er71+/− mice were mated with Mx1-Cre transgenic mice and mice with the floxed Er71 allele were mated with Mx1-Cre; Er71+/− mice. Mx1-Cre expression was induced by intraperitoneal injection of 6- to 8-week-old animals with pI-pC (20 μg/kg, Sigma, St. Louis, MO, www. sigmaaldrich.com) seven times on alternate days. Congenic (CD45.1+) mice for bone marrow (BM) transplantation experiments were obtained from The Jackson Laboratory. At 6–8 weeks of age, Mx1-Cre;Er71fl/– or Er71fl/+ mice were injected with pI-pC to generate Mx1;Er71 cKO and control mice, respectively [25].

Genotyping

Genotyping of mice was performed by polymerase chain reaction (PCR) with Taq polymerase and the primers Er71-4F (5′-ACA GCT ACA TTT TCA AGG CC-3′), Er71-5R (5′-GTC CGA GGT GTT GCA TCC C-3′), pGK-3 (5′-GCA CGA GAC TAG TGA GAC GTG CTA C-3′), and Er71-7R (5′-CCT TGA CAG GTC GAG TAG G-3′). The amplification protocol included an initial incubation at 94°C for 5 minutes followed by 30 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 65°C, and elongation for 30 seconds at 72°C. The sizes of the PCR products are 200 bp for the wild-type allele, 400 bp for the fl-neo allele, 260 bp for the floxed allele, 300 bp for the mutant allele, and 500 bp for the excised allele.

Transplantation Assays

BM transplantation was used to test the self-renewal capacity of Er71-deficient HSCs. Recipient C57BL/J mice (CD45.1+) were subjected to 9.5 Gy of whole body irradiation. For competitive transplants, CD45.1+ competitor cells (1 × 106) from wild-type C57BL/J mice were mixed with CD45.2+ donor cells (1 × 106) from Mx1-Cre;Er71fl/– or Er71fl/+ mice and were then injected into the retro-orbital vein of recipients. Deletion of Er71 in Mx1-Cre;Er71fl/– donor cells (to generate Mx1;Er71 cKO cells) was induced by seven injections of pI-pC beginning 4 weeks after transplantation. The recipient mice were analyzed for donor cells at the indicated times after the last pI-pC injection. The contribution of CD45.2+ donor cells to peripheral blood (PB) was determined with PB-derived leukocytes previously warmed under a heat lamp.

For secondary BM transplantation, recipient mice were killed 20 weeks after the first transplant, and donor cells (1 × 106) were transplanted by injection into the retro-orbital vein of irradiated recipient mice.

Flow Cytometry

Stained cells were analyzed with an LSRII, FACSAriaII, or FACSCalibur flow cytometer. Cell sorting was performed with a FACSAriaII instrument (Becton Dickinson, Mountain View, CA). Data acquisition and analysis were performed with Cell Quest Pro or Diva software (BD Biosciences, San Jose, CA, www.bdbiosciences.com) and with FlowJo software (Tree Star, Ashland, OR, www.treestar.com), respectively. BM cell suspensions were obtained by flushing of femurs and tibiae with staining buffer, and leukocytes were isolated from PB. Staining of cells for fluorescence-activated cell sorting (FACS) or flow cytometric analysis was performed in Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, www.invitrogen.com) supplemented with 2% fetal bovine serum and 1% penicillin streptomycin (staining buffer), and with conjugated monoclonal antibodies obtained from BD Biosciences or eBioscience [26]. For measurement of LinSca1+c-Kit+ (LSK) cells, BM cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34, phycoerythrin (PE)-conjugated anti-Flk2, PE- and Cy7-conjugated anti-Sca1, allophycocyanin (APC)-conjugated anti-c-Kit, and biotin-conjugated antibodies to lineage markers (CD4, CD8α, B220, Gr1, Mac1, and Ter119), the latter of which were visualized with APC- and Cy7-conjugated streptavidin. For measurement of progenitors, BM cells were stained with FITC-conjugated anti-CD34, PE-conjugated anti-CD16/32, PE-conjugated anti- IL-7Rα, APC-conjugated anti-c-Kit, PE- and Cy7-conjugated anti-Sca1, as well as biotin-conjugated antibodies to lineage markers and APC- and Cy7-conjugated streptavidin. Additional staining was performed with FITC-conjugated anti-CD45.1, PE-conjugated anti-CD45.2, FITC-conjugated anti-CD3, peridinin chlorophyll protein complex (PerCP)- and Cy5.5-conjugated anti-CD4, PE-conjugated anti-CD8α, either PerCP- and Cy5.5-conjugated or FITC-conjugated anti-B220, FITC-conjugated anti-immunoglobulin M (IgM), FITC-conjugated anti-Mac1, PE-conjugated anti-Gr1, FITC-conjugated anti-Ter119, PE-conjugated anti-CD41, and PE-conjugated anti-Tie2. For annexin V staining, freshly isolated BM cells were stained with the appropriate monoclonal antibodies, washed in binding buffer, and incubated with FITC-conjugated annexin V (BD PharMingen, San Diego, CA, www.bdbiosciences.com). For cell cycle analysis, mice were injected intraperitoneally with 1 mg of bromodeoxyuridine (BrdU; Sigma, St. Louis, MO, www.sigmaaldrich.com) per 6 g of body weight. At 19 hours after the injection, BM cells were isolated, fixed, permeabilized, and stained with FITC-conjugated anti-BrdU with the use of a staining kit (BD PharMingen, San Diego, CA, www.bdbiosciences.com). Analysis of cells in G0 was performed as described [27–29]. The LSK population was fixed, permeabilized, and stained with the use of the BrdU staining kit (BD PharMingen, San Diego, CA, www.bdbiosciences.com). The cells were then further stained with Hoechst 33342 and subjected to immunostaining with anti-Ki67 or an isotype control (FITC Ki67 staining kit, BD PharMingen, San Diego, CA, www.bdbiosciences.com). Cells were finally analyzed with a FACSAriaII instrument.

Colony-Forming Cell and Colony-Forming Unit-S Assays

For colony-forming cell assays, BM cells (1 × 104) were plated in triplicate in cytokine-supplemented methylcellulose medium (Stem Cell Tech, Vancouver, BC, Canada, www.stemcell.com). For colony-forming unit (CFU)-S assays, C57BL/J mice were used as recipients and received 9.5 Gy of radiation on the day of transplantation.

Reverse Transcription PCR and Quantitative Reverse Transcription PCR

Isolation of total RNA and cDNA synthesis were performed as described previously [18]. Briefly, total RNAs were extracted from FACS purified subsets of WT BM cells (whole BM [WBM], L, LSK, and LinSca1c-Kit+ [LK] cells), PB leukocytes from Mx1;Er71 cKO and control mice, and transfectant Er71 in MS1 ECs using Trizol reagent (Intron, Sungnam, Korea, shop.intronbio.com) according to the manufacture's instruction. Aliqouts of 1 μg of RNA were used as template for reverse transcription (RT) with oligo-dT primers (Intron). Quantitative (q) RT-PCR was performed with SYBR Green Mix (GeNet Bio, Nonsan, Korea, www.genebio.com) and an iQ5 instrument (Bio-Rad), and data were normalized by the abundance of β-actin mRNA. The normalized Ct values were measured by using the 2(−ΔCt) calculation method. Sequences of specific primers are available on request.

Microarray Analysis

All the microarray experiments were done using tools contained in the ebiogen (Republic of Korea). We sorted LSK and LK cells from pools of control and Mx1;Er71 cKO mice (n = 15). RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, www.invitrogen.com) and subjected to two rounds of amplification with WTA1 kit (Sigma, St. Louis, MO, www.sigmaaldrich.com). Amplified RNA was hybridized to Agilent 44K mouse genome array. Arrays were scanned with DNA microarray scanner (Agilent, Santa Clara, CA, www.genomics.agilent. com) running Feature Extraction Software. Raw expression values were normalized using GeneSpring GX software (Agilent, Santa Clara, CA, www.genomics.agilent.com). The threshold for considering genes differentially expressed was twofold differences. The categorization of genes into functional groupings was based on the Gene Ontology Classification System [30].

Cloning of Mouse Tie2 Promoter and Reporter Assays

The PCR-amplified mouse Tie2 promoter (−153 to +323) fragment from 129/Sv mouse ES cell genomic DNA was inserted into the XhoI and HindIII sites of pGL3-Basic and verified by sequencing. 293T cells (1 × 105 per well of a 6-well plate) were transfected with Er71 expression plasmid (pCS3-Myc-ER71), pGL3-mTie2 promoter constructs and pRL-CMV by CaCl2 precipitation. Forty-eight hours later, cells were harvested and luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega, Madison, WI, www.promega.com) according to the manufacturer's instructions. Firefly luciferase values were divided by Renilla luciferase values to calculate transfection efficiency. Data are means ± SEM from six independent experiments.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Expression of Er71 in HSCs and Effect of Haploinsufficiency of Er71 on Hematopoietic Compartments

Er71-deficient mice fail to develop blood and vascular structures during embryonic development [18]. In addition, such mice do not appear to develop hematopoietic cells, suggesting that Er71 plays a role in hematopoiesis. We, therefore, first determined the level of Er71 expression in HSCs isolated from BM of adult mice. Er71 was expressed at a high level in HSC-enriched LSK cells (Fig. 1A) and at lower levels in whole BM, Lineage (L) cells, and LK myeloid progenitor (MP) cells. This finding, together with the absence of hematopoietic cells in Er71−/− embryos, suggested that Er71 might function in HSC compartments. We, therefore, next analyzed HSCs and progenitor cells in Er71+/− mice [18]. The number of LSK cells in young (6–8 weeks) Er71+/− mice was slightly (37%) reduced compared with that in Er71+/+ animals, whereas the number of LK cells did not differ substantially between the two genotypes (Fig. 1B). Together, these results thus indicated that Er71 is preferentially expressed in HSCs and that haploinsufficiency of Er71 sustains HSC number.

thumbnail image

Figure 1. Er71 is enriched in hematopoietic stem cells (HSCs) and is haploinsufficient for sustaining HSC number. (A): Subsets of bone marrow (BM) cells (WBM, Lineage [L], LSK, and LK cells) were purified by fluorescence-activated cell sorting and assayed for Er71 mRNA by quantitative reverse transcription polymerase chain reaction analysis. Expression of Er71 in testis was examined as a control. (B): Numbers of LSK and LK cells in BM individual young (6–8 weeks) or old (92–116 weeks) Er71+/+ or Er71+/− mice (*, p < .05; n ≥ 6). Abbreviations: LK, LinSca1c-Kit+; LSK, LinSca1+c-Kit+, WBM, whole bone marrow.

Download figure to PowerPoint

Generation of Er71 Conditional Mutant Mice

To verify the role of Er71 in adult hematopoiesis, we newly generated a Cre-dependent mouse model of conditional Er71 deletion (Fig. 2A–2C). Mx1-Cre;Er71fl/– mice harbor “floxed” and null alleles of Er71 as well as a transgene for Cre recombinase that is under the control of the myxovirus resistance 1 (Mx1) gene promoter and is induced by interferon-α (via stimulation with pI-pC) (Fig. 2D) [31]. The corresponding Er71fl/+ mice were studied as controls. Treatment of Mx1-Cre;Er71fl/– mice with pI-pC resulted in efficient deletion of the floxed Er71 allele in the hematopoietic system and the consequent generation of Er71 conditional knockout (Mx1;Er71 cKO) mice (Fig. 2E). Er71 mRNA was virtually undetectable in PB leukocytes of Mx1;Er71 cKO mice (Fig. 2F), also indicative of the specific deletion of Er71 in hematopoietic tissues.

thumbnail image

Figure 2. Conditional deletion of Er71 in hematopoietic compartments. (A): Targeting strategy for conditional knockout of Er71. Blue triangles, loxP sites; cyan ovals, FLP recognition sites. (B): Southern blot analysis of EcoRI-digested genomic DNA from targeted ESCs and mice with the 5′ and 3′ probes indicated in (A). (C): Genotype polymerase chain reaction (PCR) analysis of targeted ESCs and mice. (D): Experimental scheme. (E): PCR analysis of genomic DNA extracted from the tail, PB leukocytes, BM, or Sp of Mx1;Er71 cKO and control mice for detection of the WT, mut, floxed, or excised alleles of Er71 as well as the Cre transgene. (F): Reverse transcription polymerase chain reaction (RT-PCR; upper panel) and quantitative RT-PCR (lower panel) analysis of Er71 mRNA in PB leukocytes from Mx1;Er71 cKO and control mice. β-actin mRNA was also analyzed as a control. Abbreviations: BM, bone marrow; DT, diphtheria toxin A gene; PB, peripheral blood; RI, EcoRI site; Sp, spleen. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

Effects of Er71 Deletion on HSCs

Our initial analysis revealed a slight decrease in the number of leukocytes in PB of Mx1;Er71 cKO mice compared with that in control mice (Fig. 3A). Cell number in BM and the spleen, but not that in the thymus, was also reduced in Mx1;Er71 cKO mice. We next examined the impact of conditional deletion of Er71 on HSC compartments. Multicolor FACS analysis of BM cells from Mx1;Er71 cKO mice revealed a marked decrease in the size of the LSK compartment, including both long-term (LT-HSCs: Flk2CD34LSK) and short-term (ST-HSCs: Flk2CD34+LSK) HSCs (Fig. 3B and Supporting Information Fig. 1A), suggesting that inactivation of Er71 has a pronounced effect on the size of HSC compartments at the early stage of hematopoietic development and that the reduced number of LT-HSCs might contribute to the reduction in the size of the progenitor cell population. We then sought to determine whether the reduced number of HSCs and progenitor cells in Mx1;Er71 cKO mice results from a defect in cell proliferation or apoptosis. We measured the extent of cell death in HSC and progenitor cells by staining with annexin V and propidium iodide (PI). The number of annexin V+PILSK cells was significantly increased in Mx1;Er71 cKO mice, whereas the numbers of apoptotic LK and L+ cells were unchanged (Fig. 3C and Supporting Information Fig. 1B), suggesting that apoptosis was selectively increased in LSK cells. Pulse labeling of HSC and progenitor cells with BrdU revealed a slight decrease in BrdU incorporation in LSK cells of Mx1;Er71 cKO mice compared with that for LSK cells of control animals, but no such difference was apparent for LK cells between the two genotypes (Fig. 3C and Supporting Information Fig. 1C). However, staining with Hoechst 33342 and for Ki67 revealed no marked difference in the proportion of quiescent (G0) LSK cells between the two genotypes (Fig. 3C and Supporting Information Fig. 1D). Collectively, these data suggested that increased cell death largely accounts for the reduced number of LSK cells in Mx1;Er71 cKO mice.

thumbnail image

Figure 3. Analyses of Er71-deficient hematopoietic stem cells (HSCs). (A): Total number of leukocytes in PB or of cells in BM, spleen, and thymus for Mx1;Er71 cKO and control mice 4–6 weeks after pI-pC administration (*, p < .05; n ≥ 4). (B): Total number of LSK, LT-HSC, and ST-HSC cells in individual Mx1;Er71 cKO and control mice (*, p < .05; **, p < .01, n ≥ 5). (C): Percentages of annexin V+PI cells among LSK, LK, and L+ subsets of Mx1;Er71 cKO and control mice (left panel), of BrdU incorporation in LSK and LK cells (middle panel), and of LSK cells in G0, G1, or S-G2-M phases of the cell cycle (right panel; *, p < .05; n = 4). Abbreviations: BM, bone marrow; BrdU, bromodeoxyuridine; LK, LinSca1c-Kit+; LSK, LinSca1+c-Kit+; LT-HSC, Flk2CD34LSK; PB, peripheral blood; ST-HSC, Flk2CD34+LSK.

Download figure to PowerPoint

Conditional Deletion of Er71 Affects the Differentiation of Progenitor Cells

Evidence suggests that Er71 is a key factor not only for the initiation of vasculogenesis but also for the induction of myelopoiesis in zebrafish and Xenopus embryos [19, 21, 24]. We, therefore, determined whether differentiated lineage commitment within hematopoietic compartments, including PB, BM, spleen, and thymus, is affected by deletion of Er71. The number of myeloid (Mac1+) cells was slightly decreased in the BM and PB of Mx1;Er71 cKO mice compared with that in control animals (Fig. 4A and Supporting Information Fig. 2A). Furthermore, FACS analysis of BM revealed a significant decrease in the granulocyte-monocyte progenitor (GMP) subpopulation in Mx1;Er71 cKO mice (Fig. 4B and Supporting Information Fig. 2B).

thumbnail image

Figure 4. Physiological differentiation of Er71-deficient progenitors. (A): Total number of cells of the indicated phenotypes as determined by fluorescence-activated cell sorting analysis of PB, BM, spleen, and thymus from Mx1;Er71 cKO and control mice (n ≥ 4). (B): Total number of progenitors including CMP (CD34+CD16/32LK), MEP (CD34CD16/32LK), GMP (CD34+CD16/32+LK), and CLP (IL-7Rα+LinSca1loc-Kitlo) cells in BM of individual Mx1;Er71 cKO and control mice (*, p < .05; n = 5). (C): Number of differentiated colonies and morphological analysis for a colony-forming cell assay with BM cells from Mx1;Er71 cKO and control mice. Cells (1 × 104) were plated in cytokine-supplemented methylcellulose medium in triplicate (**, p < .01; n = 10). (D): Number of CFU-S8 and CFU-S12 in individual recipient mice receiving BM cells (1 × 105) from Mx1;Er71 cKO or control mice (n ≥ 3). Abbreviations: BM, bone marrow; CFU, colony-forming unit; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythroid progenitor; PB, peripheral blood.

Download figure to PowerPoint

To explore further the functionality of Er71-deficient HSC and progenitor cells, we performed in vitro methylcellulose assays with BM cells in the presence of cytokines. The total number of colonies obtained was significantly reduced for Mx1;Er71 cKO mice compared with that for control mice, mainly as a result of a decreased number of myeloid-lineage cells (Fig. 4C). Colony-specific PCR analysis revealed the complete deletion of Er71 alleles in all surviving colonies derived from Er71-deficient stem and progenitor cells (Supporting Information Fig. 2C and 2D). We next determined the impact of Er71 deletion on the short-term differentiation activity of stem and progenitor cells in vivo. To this end, we scored colony-forming units-spleen at 8 and 12 days (CFU-S8 and −S12) after transplanting BM cells from control or Mx1;Er71 cKO mice into lethally irradiated WT mice. As previously shown [25], stem and progenitor cells from BM of control mice formed colonies in the spleen of the recipient animals (Fig. 4D and Supporting Information Fig. 2E). However, the numbers of both CFU-S8 and CFU-S12 for BM cells of Mx1;Er71 cKO mice were decreased compared with those for control mice, indicating that Er71 is required for the short-term differentiation activity of circulating stem and progenitor cells.

Deletion of Er71 Reduces HSC Repopulation Capacity

To address whether the defects in Er71-deficient HSCs are cell autonomous, we mixed equal numbers of CD45.2+ BM cells from control or Mx1-Cre;Er71fl/– mice with CD45.1+ BM cells from WT mice and transplanted the mixed cell population into irradiated recipient animals (Fig. 5A). Deletion of the floxed Er71 allele in donor cells was induced by pI-pC injection beginning 4 weeks after transplantation. Subsequent analysis of PB of the recipients for donor cells showed that the number of Mx1;Er71 cKO cells was smaller than that of control cells (Fig. 5B). PCR analysis of genomic DNA isolated from PB or BM of recipients transplanted with Mx1-Cre;Er71fl/– cells confirmed complete excision of the floxed Er71 allele after pI-pC treatment (Fig. 5C and 5E), indicating that hematopoietic reconstitution was mediated by Mx1;Er71 cKO donor cells. Analysis of the donor-derived BM compartment of recipients at 20 weeks after transplantation also revealed that the frequency of total or LSK cells as well as the ability of HSCs to generate mature Mac1+ cells were reduced for Mx1;Er71 cKO cells compared with control cells (Fig. 5D). In vitro assays also showed that the contribution of Mx1;Er71 cKO HSCs to the myeloid lineage was more markedly affected than was that to lymphoid lineages (Supporting Information Fig. 3A). These data thus underlined the importance of Er71 in HSCs, with a selective defect also being apparent in the ability of Er71-deficient HSCs to generate myeloid cells. To further explore the long-term repopulation of Mx1; Er71 cKO HSCs, we isolated donor BM cells at 20 weeks after the first transplant, and injected them into secondary recipient mice. Control donor cells were able to reconstitute the secondary recipients efficiently, whereas secondary Mx1; Er71 cKO donor cells failed to reconstitute the secondary recipients and died within 4 weeks after secondary transplant, suggesting that the activity of HSC was reduced following Er71 deletion (Supporting Information Fig. 3B). Together, these data were indicative of a cell-autonomous requirement for Er71 in HSCs.

thumbnail image

Figure 5. Er71 maintains HSCs in a cell-autonomous manner. (A): Experimental scheme for BMT experiments. (B): The percentage of PB leukocytes of recipients derived from control (Er71fl/+) or Mx1-Cre;Er71fl/– cells was determined by fluorescence-activated cell sorting (FACS) at the indicated times (n ≥ 4). (C): Representative polymerase chain reaction (PCR) analysis of Er71 alleles with genomic DNA of PB leukocytes from recipients of competitive transplants at 16 weeks after transplantation. (D): FACS analysis of the percentage contributions of Mx1;Er71 cKO or control cells of the indicated phenotypes in the BM of recipients at 20 weeks post-transplantation (*, p < .05; **, p < .01; n = 4). (E): Representative PCR analysis of Er71 alleles with genomic DNA of BM cells from recipients of competitive transplants at 20 weeks after transplantation. Abbreviations: BM, bone marrow; BMT, bone marrow transplantation; LSK, LinSca1+c-Kit+; PB, peripheral blood; WT, wild type.

Download figure to PowerPoint

Er71 Activates Tie2 Expression

We next performed global gene expression profiling analyses to identify Er71-dependent genes and pathways involved in the regulation of HSC compartments. For these studies, LSK and LK MP cells were prospectively sorted from BM harvested 4 weeks after the last injection of pI-pC. Analysis of gene expression data from LSK cells revealed 1,520 upregulated genes and 2,025 downregulated genes in Er71 mutants (Supporting Information Fig. 4A and 4B and Supporting Information Table 1). We generated gene signatures specific for HSCs by subtracting the genes expressed in LSK cells (Fig. 6A). Of the HSC signature genes, 53% (72 of 137) of these selected genes were significantly deregulated in Er71-deficient LSK cells. We also found that many genes belonging to HSC annotation groups, such as genes involved in the regulation of HSCs (Pbx1, Bmi1, Egr1), surface receptors (Tie2 [also known as Tek], Flt3), and regulators of HSC survival (Mcl1), were downregulated in Er71-deficient LSK cells. Real-time qRT-PCR analysis further confirmed that expression of Tie2, Bmi1, and Flt3 genes were decreased in purified LSK cells (Fig. 6B).

thumbnail image

Figure 6. Ablation of Er71 suppresses Tie2 expression in hematopoietic stem cells (HSCs). (A): HSC regulation-associated genes are deregulated in Er71-deficient LSK cells. The heat map shows the genes associated with the gene ontology of the biological process termed “hematopoietic stem cell.” (B): Representative quantitative reverse transcription polymerase chain reaction analysis of Tie2, Bmi1, Flt3, and Pbx1 mRNA in LSK cells of Mx1;Er71 cKO mice relative to that in those of control mice (n = 6). (C): Fluorescence-activated cell sorting analysis of Tie2 expression on LSK or LK cells isolated from Mx1;Er71 cKO and control mice (left panel). MFI values for Tie2 are shown in the right panel (**, p < .01; n = 5). (D): Model for the role of Er71 in HSC and progenitor cell maintenance. Blue boxes include cells affected by Er71 loss. Dotted lines indicate Er71 binds to the promoter of Tie2 and activates its expression. Abbreviations: CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; LK, LinSca1c-Kit+; LSK, LinSca1+c-Kit+; LT-HSC, Flk2CD34LSK; MEP, megakaryocyte-erythroid progenitor; MFI, Mean fluorescence intensity; MP, myeloid progenitor; PE, phycoerythrin; ST-HSC, Flk2CD34+LSK. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

Among the 72 deregulated genes, it was particularly notable that Tie2 is downregulated in Er71-deficient HSCs. The Tie2 gene was previously reported to be a direct downstream target of Er71 [20], with its promoter-enhancer region shown to contain Ets binding sites [32]. Tie2 is also expressed in HSCs and contributes to the maintenance and survival of these cells [33–35]. These observations prompted us to further test whether Er71 might regulate Tie2 expression in HSCs. Consistently, the amounts of Tie2 protein (Fig. 6C) were decreased in LSK cells of Mx1;Er71 cKO mice compared with those in control mice. However, there were no substantial differences in Tie2 expression at the protein level in LK cells between the two genotypes. These results thus suggested that Tie2 expression is at least in part regulated by Er71 in LSK cells. To confirm that the activation of Tie2 expression by Er71 occurs at the transcriptional level, we also measured the activity of a luciferase reporter construct containing −153 to +323 of the Tie2 promoter region (Supporting Information Fig. 4C). Cotransfection of 293T cells with an expression vector for Er71 resulted in a marked increase in the expression of the reporter gene (Supporting Information Fig. 4D). Forced expression of Er71 was also found to increase endogenous Tie2 expression in endothelial cells (Supporting Information Fig. 4E). Together, these data suggested that Er71 contributes to HSC maintenance by inducing Tie2 transcription.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Er71 functions as a transcription factor that initiates vasculogenesis in mammals, and its ortholog (Etsrp) in zebrafish and Xenopus regulates the induction of myelopoiesis [19, 21, 22]. We have now shown that Er71 functions as a regulator of HSC activity and regulates myeloid differentiation in a cell-autonomous manner (Fig. 6D). Inactivation of Er71 in mice was thus found to affect the primitive hematopoietic compartment including HSCs. This defect with loss of Er71 is likely a consequence of an increased level of apoptosis. We also found that hematopoiesis was impaired when Er71 was deleted in donor cells after BM transplantation into irradiated recipient animals. This phenotype was characterized by aberrant reconstitution of HSCs, which showed a reduced ability to generate myeloid cells. Our present findings therefore implicate Er71 as an intrinsic regulator of HSCs. Although other mature hematopoietic lineages were not affected, myeloid differentiation was impaired after deletion of Er71 induced by pI-pC administration. The reduction in the number of mature myeloid cells associated with Er71 deficiency was attributable to a block in the transition from common myeloid progenitors to GMPs.

Er71 contributes to the efficient expression of Flk1, Tal1, Mef2c, Pecam1, and Tie2 during mouse embryonic development. Among these Er71 target genes, Tie2, which encodes a receptor tyrosine kinase, plays a key role in definitive hematopoiesis [36–38] and is expressed preferentially in HSCs [33, 34]. HSCs expressing Tie2 are quiescent and resistant to the induction of apoptosis, and they maintain long-term repopulating activity in vivo through interaction with the Tie2 ligand angiopoietin 1 [35]. Tie2 signaling is important for the maintenance and survival of HSCs in adult BM. The Tie family of proteins has been shown to be required for postnatal hematopoiesis in a mouse model [39]. Adult Tie1−/−Tie2−/− chimeric mice thus fail to maintain HSCs in the hematopoietic microenvironment. However, the transcription factors that activate Tie2 expression to maintain HSCs in adults have remained unknown. We have now found that the expression of Tie2 in HSCs of adult mice is downregulated as a result of Er71 deletion. Consistent with the recent observation that Tie2 is a direct downstream target of Er71, we also found that Er71 activates the Tie2 promoter. The downregulation of Tie2 expression induced by deletion of Er71 may trigger apoptosis in HSCs, resulting in a decrease in HSC number. Several Ets transcription factors, including PU.1, Tel, Ets1, and Fli1, have been shown to play central roles in adult hematopoiesis [5, 6]. Among these Ets transcription factors, no single transcription factor has previously been shown to control HSC activity by regulating Tie2 expression.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Ets transcription factor Er71 is an important regulator of HSC maintenance by regulating Tie2 expression in adult mice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This study was supported by grants from the National Creative Research Initiatives Program (2010-0018277), World Class University Program of the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (R31-2008-000-10071-0).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional supporting information available online.

FilenameFormatSizeDescription
STEM_597_sm_suppinfo.pdf3150KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.