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Marking of definitive HSC precursors in E7.5–E8.5 embryos using an Abcg2-CreER lineage-tracing mouse model

  • Author Footnotes
    1 SF and SZ contributed equally to this work.
    Soghra Fatima
    Footnotes
    1 SF and SZ contributed equally to this work.
    Affiliations
    Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
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  • Author Footnotes
    1 SF and SZ contributed equally to this work.
    Sheng Zhou
    Correspondence
    Offprint requests to: Sheng Zhou, Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105
    Footnotes
    1 SF and SZ contributed equally to this work.
    Affiliations
    Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
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  • Brian P. Sorrentino
    Affiliations
    Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA
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  • Author Footnotes
    1 SF and SZ contributed equally to this work.
Open AccessPublished:July 02, 2018DOI:https://doi.org/10.1016/j.exphem.2018.06.286

      Highlights

      • Abcg2 expression marks precursors of hematopoietic stem cells (HSCs) at embryonic day 7.5 (E7.5)–E8.5.
      • At least 333 precursors of HSCs exist at E7.5–E8.5.
      • Abcg2 does not mark precursors of kidney proximal tubule cells, hepatocytes, and small intestine epithelial cells at E7.5–E8.5.
      Abcg2, a member of the ATP-binding cassette transporter family, is expressed in adult hematopoietic stem cells (HSCs) and is required for the side population phenotype of adult bone marrow HSCs and other adult tissue-specific stem cells. Lineage tracing in adult mice using the Abcg2-Cre mouse model showed that Abcg2 marks HSCs, intestinal stem cells, and spermatogonial stem cells. It is unclear whether definitive HSCs or their precursors in early embryonic development can be marked by Abcg2 expression. Here, we treated pregnant Abcg2 Cre/Cre RosaLSL-YFP mice with a single injection of 4-hydroxytamoxifen at embryonic day 7.5. Four months after birth, a small yellow fluorescent protein-positive (YFP+) cell population could be detected in all of the major white blood cell lineages and this was stable for 8 months. Transplant of bone marrow cells or Sca1+YFP+ cells from these mice showed continued multilineage marking in recipient mice at 4 months. These results demonstrate that Abcg2 expression marks precursors to adult long-term repopulating HSCs at E7.5 to E8.5 and contributes to a stable subpopulation of HSCs well into adulthood.
      Abcg2 is a plasma membrane transporter that is expressed in the side population cells of a variety of tissues, including cancer cells, and is required for their SP phenotype [
      • Zhou S
      • Schuetz JD
      • Bunting KD
      • Colapietro AM
      • Sampath J
      • Morris JJ
      • et al.
      The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.
      ,
      • Goodell MA
      • Brose K
      • Paradis G
      • Conner AS
      • Mulligan RC
      Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.
      ]. In adult mice, virtually all hematopoietic stem cells (HSCs) express Abcg2 [
      • Tadjali M
      • Zhou S
      • Rehg J
      • Sorrentino BP
      Prospective isolation of murine hematopoietic stem cells by expression of an Abcg2/GFP allele.
      ]. Lineage-tracing studies using an Abcg2CreERRosaYFP allele in the adult mice confirmed expression of Abcg2 in adult HSCs and revealed that adult tissue-specific intestinal stem cells and spermatogonial stem cells also express Abcg2 [
      • Fatima S
      • Zhou S
      • Sorrentino BP
      Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.
      ]. Definitive HSCs (dHSCs) have been identified at embryonic day 10.5 (E10.5) in the midgestation dorsal aorta with an estimated total of <100 of these cells [
      • Boisset JC
      • Clapes T
      • Klaus A
      • et al.
      Progressive maturation toward hematopoietic stem cells in the mouse embryo aorta.
      ]. The phenotype and number of precursors of dHSCs (pdHSCs) at earlier developmental stages at E7.5–E8.5 is less clear. We have shown that both hematopoiesis and HSC number and functions are normal in Abcg2–/– mice [
      • Zhou S
      • Morris JJ
      • Barnes Y
      • Lan L
      • Schuetz JD
      • Sorrentino BP
      Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo.
      ]. In our Abcg2CreER lineage-tracing mouse model, the ires-CreER expression cassette is inserted downstream of the stop codon of Abcg2, so Abcg2 was coexpressed with endogenous Abcg2 [
      • Fatima S
      • Zhou S
      • Sorrentino BP
      Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.
      ]. This mouse model allowed us to perform lineage tracing during embryo development under unperturbed conditions.

      Methods

      Mice

      Abcg2CreERT2RosaEYFP mice were generated previously in our laboratory [
      • Fatima S
      • Zhou S
      • Sorrentino BP
      Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.
      ]. C57BL/6J mice were purchased from The Jackson Laboratory. All experiments with mice were performed according to a protocol approved by the St. Jude Children's Research Hospital Institutional Animal Care and Use Committee.

      Tamoxifen treatment

      4-Hydroxytamoxifen (4-OHT, Millipore Sigma, USA) was dissolved in sunflower oil at a concentration of 5 mg/mL. Pregnant Abcg2CreERT2/CreERT2+ RosaEYFP/EYFP mice were treated with one intraperitoneal injection of 4-OHT at E7.5 using overnight timed breeding pairs.

      Antibody staining for flow cytometry analysis

      Expression of yellow fluorescent protein (YFP) in different peripheral blood and bone marrow cells was detected by flow cytometry. Peripheral blood samples were stained for B220, CD3, Gr1, Mac1, and Ter119 using fluorescent-conjugated antibodies (B220-PerCP-Cy5.5, CD3-APC, Gr1-APC-Cy7, Mac1-Alexa700, and Ter119-PE-Cy7, Becton Dickinson, USA).

      Organ collection and processing

      Mice were euthanized and intravenously perfused with phosphate-buffered saline followed by 2% paraformaldehyde. The organs were dissected and further fixed overnight at 4°C. Organs were cryopreserved with 30% sucrose and embedded in optimal cutting temperature compound (Tissue-Tek, Sakura, USA).

      Immunofluorescence microscopy

      Fourteen–micrometer-thick tissue sections prepared on a cryostat were immunostained with antibodies specific for green fluorescent protein (GFP, A11122 rabbit IgG, Invitrogen, USA), Pecam (BD 553070, rat IgG, Becton Dickinson, USA), and Abcg2 (clone BXP53 MC-981, Kamiya Biomedical Company, USA). For Pecam and Abcg2 antibodies, a secondary Alexa Fluor 488 donkey anti-rat antibody was used (A21208, Invitrogen, USA) and, for GFP antibody, a secondary Cy3 donkey anti-rabbit antibody was used (AP18C, Millipore, USA). Images were captured with a confocal laser-scanning microscope (Zeiss).

      Transplantation

      Sca1+ cells were first enriched from bone marrow of selected mice that were treated with 4-OHT at E7.5 and were 9 months of age at that time. YFP+ cells were then sorted and transplanted along with Sca1 cells into lethally irradiated (1100 rad) C57BL/6J recipient mice. Bone marrow nucleated cells were also transplanted from one donor mouse.

      Results and discussion

      Single pulse treatment of mice with 4-OHT at E7.5 marks pdHSCs

      In our lineage-tracing mouse model, the CreERT2 was coexpressed with endogenous Abcg2 because the Ires-CreERT2 expression cassette is inserted downstream of the Abcg2 coding sequence [
      • Fatima S
      • Zhou S
      • Sorrentino BP
      Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.
      ]. Upon exposure to 4-OHT, the Cre translocates to the nucleus and deletes the stop element upstream of the EYFP transgene, which leads to ubiquitous, permanent expression of YFP in all progenies. We have also shown that HSC development was normal in the Abcg2–/– mice [
      • Zhou S
      • Morris JJ
      • Barnes Y
      • Lan L
      • Schuetz JD
      • Sorrentino BP
      Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo.
      ], so hematopoietic development is most likely unperturbed in the Abcg2CreERT2RosaEYFP mouse model. To limit the length of exposure of 4-OHT to a stringent short time window, we treated pregnant homozygous Abcg2CreERT2RosaEYFP females with a single 1 mg injection of 4-OHT at E7.5. It has been shown that this treatment does not mark cells beyond 24 hours due to the short half-life of 4-OHT [
      • Robinson SP
      • Langan-Fahey SM
      • Johnson DA
      • Jordan VC
      Metabolites, pharmacodynamics, and pharmacokinetics of tamoxifen in rats and mice compared to the breast cancer patient.
      ,
      • Zovein AC
      • Hofmann JJ
      • Lynch M
      • et al.
      Fate tracing reveals the endothelial origin of hematopoietic stem cells.
      ]. A total of 18 live pups were born from three dams. The YFP marking in white blood cells was 0–6.2% at 1 month, 0.1–4.5% at 4 months, and 0–3.5% at 8 months (Figures 1A and 1B). In the majority of mice, the YFP marking was relatively stable between 1 and 8 months (Figures 1A and 1B). The YFP marking occurred in the CD3+, B220+, Gr1+, and Mac1+ lineages, suggesting pdHSC marking. None of the mice not exposed to 4-OHT had any YFP expression in the peripheral blood cells (Figure 1C, lower panels).
      Fig 1
      Figure 1Abcg2 expression marks E7.5–E8.5 pdHSCs. Pregnant Abcg2CreERT2RosaEYFP mice were treated with a single injection of 4-OHT at E7.5. Fetuses were allowed to be born and grow to adulthood. Peripheral blood was taken at various time points and analyzed for YFP expression in the white blood cell lineages. Mice that had >1% YFP+ white blood cells at 1 month are graphed in (A) and mice that had <1% YFP+ white blood cells at 1 month are graphed in (B). (C) YFP expression in white blood cell lineages from a representative mouse. One Abcg2CreERT2RosaEYFP mouse that did not receive 4-OHT showed absolutely no YFP expression ((C), lower panels). Total bone marrow cells (D) or sorted Sca1+YFP+ cells (E) from mice treated with 4-OHT at E7.5 at 9 months of agewere transplanted into lethally irradiated recipient mice and YFP expression was analyzed in peripheral blood cell lineages 4 months after transplantation. (F) YFP expression in lineages from one representative recipient mouse.
      Bone marrow cells from mouse #2888, which had YFP marking in the peripheral blood, bone marrow, spleen, and thymus of 1.7%, 1.3%, 1.5%, and 2%, respectively, at 9 months were transplanted into four recipient mice at a dose of 7.5 × 106 cells each. Four months after the transplantation, all four mice had similar or higher YFP+ cells in the peripheral blood compared with the donor bone marrow (Figure 1D). The YFP marking in a second donor (mouse #2887) in the peripheral blood, bone marrow, spleen, and thymus was 1.6%, 1.7%, 1.3%, and 0.8%, respectively. A total of 15,342 sorted Sca1+YFP+ cells were mixed with an equal number of sorted Sca1+YFP cells, along with 2 × 105 sorted Sca1 cells and transplanted into each of three recipient mice. Four months after the transplantation, >59% of cells in all lineages in peripheral blood were marked by YFP expression in all three recipient mice (Figures 1E and 1F). The third mouse (#2873) had YFP marking in the peripheral blood, bone marrow, spleen, and thymus of 0.9%, 0.4%, 0.8%, and 0.8%, respectively. A total of 2800 sorted Sca1+YFP+ cells were mixed with 2800 sorted Sca1+YFP cells, along with 2 × 105 Sca1 cells, and transplanted into two recipient mice. Four months later, the YFP marking in the peripheral blood mononuclear cells was 13.4% and 88.4%. These results suggest that Abcg2 is expressed in pdHSCs at E7.5–E8.5. Immunofluorescence staining of E7.5 embryo sections showed that Abcg2 is expressed primarily in the visceral endoderm, but lower expression can also be seen in some mesoderm cells (Supplementary Figure E1, online only, available at www.exphem.org). In a study using the Runx1WT/CreER mouse line, 1–10% of marking in all adult lineages were seen when mice were treated with 4-OHT at E7.5, which was interpreted as a contribution of yolk sac cells to adult hematopoiesis [
      • Samokhvalov IM
      • Samokhvalova NI
      • Nishikawa S
      Cell tracing shows the contribution of the yolk sac to adult haematopoiesis.
      ]. However, this interpretation is challenged by the temporal alteration in the emergence of dHSCs in heterozygous Runx1 embryos [
      • Cai Z
      • de Bruijn M
      • Ma X
      • et al.
      Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo.
      ,
      • Lux CT
      • Yoder MC
      Novel methods for determining hematopoietic stem and progenitor cell emergence in the murine yolk sac.
      ]. Our Abcg2-CreER mouse model could complement the Runx1 model in future studies because Abcg2 expression was not altered.
      The low level of marking could reflect inefficient recombination because of either relatively low levels of expression of the recombinant allele in these embryonic HSC precursors or inefficient nuclear localization with the single 4-OHT pulse. Alternatively, these marked embryonic HSC precursors may generate only a minor population of adult HSCs that compete against a larger fraction of HSCs that arise from precursors that originate later in gestation after the 24-hour 4-OHT washout [
      • Samokhvalov IM
      • Samokhvalova NI
      • Nishikawa S
      Cell tracing shows the contribution of the yolk sac to adult haematopoiesis.
      ].

      Estimation of the number of pdHSCs in early embryos

      The total number of dHSCs/RUs within the developing embryo at E12 was estimated to be ∼66 using the transplantation assay [
      • Kumaravelu P
      • Hook L
      • Morrison AM
      • et al.
      Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.
      ]. It has been difficult to measure the number of pdHSCs at E7.5–E8.5 using transplantation-based assays because these pdHSCs are not mature enough to reconstitute recipient hematopoiesis. Recent studies have also shown that HSCs could independently arise from the vitelline and umbilical arteries and vasculatures of the placenta and the brain [
      • Gordon-Keylock S
      • Sobiesiak M
      • Rybtsov S
      • Moore K
      • Medvinsky A
      Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs.
      ,
      • Gekas C
      • Dieterlen-Lievre F
      • Orkin SH
      • Mikkola HK
      The placenta is a niche for hematopoietic stem cells.
      ,
      • Li Z
      • Lan Y
      • He W
      • et al.
      Mouse embryonic head as a site for hematopoietic stem cell development.
      ]. Given that cells, including the pdHSCs are digital entities, low marking at E7.5 allows us to estimate the number of pdHSCs at this stage. In several mice, ∼0.3–0.8% of cells are marked (Figure 2). If we assume that the 0.3% YFP+ represents marking of a single pdHSC and that the lineage output capacity of all pdHSCs are similar, then we could estimate that there are at least 333 pdHSCs at this stage. Even if the 4-OHT were effective as late as E10.5, the number of pdHSCs would be estimated to be at least 333 during E7.5–E10.5. This estimation is consistent with a recent study using a multicolored lineage-tracing mouse model showing that ∼719 pdHSCs were present at E7–E8.5 [
      • Ganuza M
      • Hall T
      • Finkelstein D
      • Chabot A
      • Kang G
      • McKinney-Freeman S
      Lifelong haematopoiesis is established by hundreds of precursors throughout mammalian ontogeny.
      ].
      Fig 2
      Figure 2Low YFP marking in the blood allows estimation of the number of pdHSCs at E7.5–E8.5. Abcg2CreERT2RosaEYFP mice were treated with a single injection of 4-OHT at E7.5. Fetuses were allowed to be born and grow to adulthood. YFP expression in white blood cells at 8 months is shown for eight mice.

      Lack of marking in kidney proximal tubule epithelium, hepatocytes, and intestinal epithelium

      When adult mice were treated with 4-OHT, kidney proximal tubule cells, hepatocytes, and intestinal epithelial cells were all marked efficiently [
      • Fatima S
      • Zhou S
      • Sorrentino BP
      Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.
      ]. The kidney proximal tubules start to develop from metanephric mesenchyme around E10.5 [
      • Li X
      • Oghi KA
      • Zhang J
      • et al.
      Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis.
      ]. The hepatocytes develop from the foregut endoderm around E8.0 [
      • Zorn AM
      • Wells JM
      Vertebrate endoderm development and organ formation.
      ]. Small intestinal epithelium development starts around E9.0 from the gut tube [
      • Spence JR
      • Lauf R
      • Shroyer NF
      Vertebrate intestinal endoderm development.
      ]. When mice were treated at E7.5 and tissues analyzed 9 months after birth, no YFP marking in kidney proximal tubules and intestinal epithelium was seen (Figures 3A and 3B; n = 4). A small number of cells in the liver were marked by YFP expression (Figure 3C). Costaining with the endothelial marker Pecam showed that majority of these YFP+ cells are located in the blood vessels, showing that they are hematopoietic cells. These results prove that the precursor cells to the kidney proximal tubules, hepatocytes, and small intestine epithelial cells do not express Abcg2 at E7.5–E8.5 and that the marking in pdHSCs is specific.
      Fig 3
      Figure 3Lack of marking in kidney proximal tubules, hepatocytes, and small intestine epithelial cells. Abcg2CreERT2RosaEYFP mice were treated with a single injection of 4-OHT at E7.5. Fetuses were allowed to be born and grow to adulthood and tissue sections were stained with anti-GFP antibody, anti-Abcg2 antibody, Bxp-53, and DAPI. Shown are the kidney (A), small intestine (B), and liver (C) all at 20 × magnification.
      In summary, our Abcg2 lineage-tracing mouse model demonstrated that pdHSCs are relatively specifically marked at E7.5–E8.5 by Abcg2 expression. Therefore, this mouse model is a useful tool for studying these pdHSCs.

      Conflict of interest disclosure

      The authors declare no competing financial interests.

      Acknowledgments

      The authors thank the following shared resources at St. Jude for providing outstanding scientific support for these studies: the Flow Cytometry and Cell Sorting Core, the Transgenic/Gene Knockout Core, the Animal Resource Center, and the Veterinary Pathology Core. These facilities are supported by a grant from the National Institutes of Health Cancer Center (P30CA21765).
      This work was supported by a grant from the NIH ( R01 HL67366 to B.P.S.) and by the American Lebanese Syrian Associated Charities.

      Appendix. Supplementary materials

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