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    MMC 1

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    anower jabed
    This document provides supplemental figures and information to accompany a research article on functional reprogramming of polyploidization in megakaryocytes. Figure S1 validates a genetic tool for megakaryocyte-specific ablation and shows similar proliferation of megakaryocytes lacking Aurora B. Figure S2 shows increased hematopoietic progenitors in mice lacking Cdc20. Figure S3 depicts time-lapse microscopy of endomitosis. Figure S4 shows time-lapse of endocycling Cdk1-deficient cells. Figure S5 examines polyploidization and cell fate with combined Cdk mutations in a Cdc20-null background. Experimental procedures are also described.

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    MMC 1

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    This document provides supplemental figures and information to accompany a research article on functional reprogramming of polyploidization in megakaryocytes. Figure S1 validates a genetic tool for megakaryocyte-specific ablation and shows similar proliferation of megakaryocytes lacking Aurora B. Figure S2 shows increased hematopoietic progenitors in mice lacking Cdc20. Figure S3 depicts time-lapse microscopy of endomitosis. Figure S4 shows time-lapse of endocycling Cdk1-deficient cells. Figure S5 examines polyploidization and cell fate with combined Cdk mutations in a Cdc20-null background. Experimental procedures are also described.

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    This document provides supplemental figures and information to accompany a research article on functional reprogramming of polyploidization in megakaryocytes. Figure S1 validates a genetic tool for megakaryocyte-specific ablation and shows similar proliferation of megakaryocytes lacking Aurora B. Figure S2 shows increased hematopoietic progenitors in mice lacking Cdc20. Figure S3 depicts time-lapse microscopy of endomitosis. Figure S4 shows time-lapse of endocycling Cdk1-deficient cells. Figure S5 examines polyploidization and cell fate with combined Cdk mutations in a Cdc20-null background. Experimental procedures are also described.

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    4 views8 pages

    MMC 1

    Uploaded by

    anower jabed
    This document provides supplemental figures and information to accompany a research article on functional reprogramming of polyploidization in megakaryocytes. Figure S1 validates a genetic tool for megakaryocyte-specific ablation and shows similar proliferation of megakaryocytes lacking Aurora B. Figure S2 shows increased hematopoietic progenitors in mice lacking Cdc20. Figure S3 depicts time-lapse microscopy of endomitosis. Figure S4 shows time-lapse of endocycling Cdk1-deficient cells. Figure S5 examines polyploidization and cell fate with combined Cdk mutations in a Cdc20-null background. Experimental procedures are also described.

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    of 8

    Developmental Cell

    Supplemental Information

    Functional Reprogramming of Polyploidization


    in Megakaryocytes
    Marianna Trakala, Sara Rodríguez-Acebes, María Maroto, Catherine E. Symonds, David
    Santamaría, Sagrario Ortega, Mariano Barbacid, Juan Méndez, and Marcos Malumbres
    Supplementary Figures

    Figure S1. Genetic tools for megakaryocyte-specific ablation of cell cycle regulators (related to Figure
    1). a) Schematic representation of the LoxP-STOP-LoxP (LSL)- Katushka-fluorescent protein (KFP) reporter.
    b) Validation of Pf4-Cre activity using the LSL-KFP reporter in polyploid megakaryocytes. Bone marrow
    progenitors (lin-) carrying the Pf4-Cre and LSL-KFP alleles were stimulated in vitro in the presence of TPO and
    Cre activity was monitored by analyzing KFP. As shown in the histograms, three or four days after TPO
    treatment, most polyploid (≥8C) megakaryocytes express KFP as a consequence of the Cre-dependent
    excision of the STOP cassette, wheras less than half of the 4C cells and a very reduced percentage of 2C
    cells are positive for this marker. c) Immunodetection of Aurora B in low ploidy (2C-4C) or high ploidy (≥4C)
    megakaryocytes (CD41-positive cells) in the bone marrow from the indicated genotypes. -actin was used as
    a loading control. d) The cumulative percentage of CD41+ cells (mean ± SEM) is similar in Aurkb() and
    Aurkb() cultures (P>0.05; Student’s t-test) on days 2-5 after TPO stimulation of E13.5 fetal liver cells. e)
    Representative images of fetal liver-derived megakaryocytes 5 days after stimulation with TPO showing similar
    number and appearance of large megakaryocytes. Pro-platelet formation occurs in the absence of Aurora B as
    indicated by arrows in right lower panel. Scale bars, 50 m.
    Figure S2. High levels of hematopoietic progenitors as a consequence of severe thrombocytopenia in
    Cdc20() mice (related to Figure 2). a) Immunodetection of Cdc20 in high ploidy (≥4C) megakaryocytes
    (CD41-positive cells) in the bone marrow from the indicated genotypes. -actin was used as a loading control.
    b) Hematoxylin and eosin (H&E) staining and immunohistochemical staining for Von Willebrand Factor (VWF)
    in spleen sections from in 8-12 week-old animals with the indicated genotypes. Scale bars, 50 m. c)
    Percentage of hematopoietic progenitors from the bone marrow of Cdc20() and control in 8-12 week-old
    mice after FACS analysis with specific markers (see Extended Experimental Protocols). HSCs, Hematopoetic
    stem cells; CLPs, common lympoid progenitors; CMPs, Common myeloid progenitors; MEPs, megakaryocyte-
    erythrocyte progenitors; GMPs, granulocyte-macrophage progenitors. n=5 mice per group d) Representative
    data and quantification of the CD41+ population after FACS analysis of total fetal liver (FL) cells from E12.5
    embryos or cells from the bone marrow (BM) of 8 week-old mice. n=3 mice per group. All graphs show mean ±
    SEM. Statistics were performed by Student’s t-test; ns, not significant; **, p<0.01; ***, p<0.001.
    Figure S3. Time-lapse microscopy of endomitotic polyploidization in vitro (related to Figure 4). Time-
    lapse microscopy of bone marrow derived megakaryocytes stably expressing histone-GFP (H2B-GFP; green),
    lamin-CFP (blue) and geminin-mCherry (Red). a) Representative pictures of an individual cell during the G1-S
    transition. Time=0 represents the exit from endomitosis characterized by degradation of geminin (red),
    decondensation of chromatin (green) and nuclear envelope reformation (blue). During G1-phase cells are
    negative for geminin and start to express it upon G1-S transition. During S-phase the increase in nuclear
    volume can be appreciated. b) Representative images of an individual cell during endomitosis. Endomitosis is
    characterized by chromatin condensation and the absence of nuclear envelope, resulting in the pancellular
    distribution of geminin, which is exclusively nuclear during interphase. In late stages of endomitosis, geminin is
    degraded and nuclear envelope reforms and chromatin decondenses. c) Plots showing the relative units of
    geminin-mCherry mean fluorescent intensity, H2B-GFP mean fluorescent intensity, and nuclear and cellular
    volume during the endomitotic cell cycle in a representative megakaryocyte. The purple frame depicts
    endomitosis (M).
    Figure S4. Time-lapse microscopy of endocycling Cdk1-deficient cells in vitro (related to Figure 4). a)
    Time-lapse microscopy of bone marrow derived Cdk1() megakaryocytes stably expressing histone-GFP
    (H2B-GFP; green) and geminin-mCherry (Red). Time=0 was arbitrarily set. Geminin is degraded (at ~17-18 h)
    in the absence of mitosis; i.e. without nuclear envelope breakdown (as detected by the absence of nuclear-to-
    cytoplasmic redistribution of geminin, and absence of DNA condensation). During G1 cells are negative for
    geminin and start to express it at the G1-S transition. b) Plots showing the relative units of geminin-mCherry
    mean fluorescent intensity, H2B-GFP mean fluorescent intensity, and nuclear and cellular volume during the
    endoreplication process in a representative Cdk1() megakaryocyte. c) Time-lapse microscopy of
    megakaryocytes expressing geminin (green), lamin (blue) and KFP (red). d) Immunodetection of Cdk1 in
    CD41-positive cells isolated from the bone marrow of Pf4-Cre; Cdk1(/); LSL-KFP or Pf4-Cre; Cdk1(/);
    LSL-KFP animals. KFP-positive (+) or negative (-) cells were isolated by flow cytometry. -actin was used as a
    loading control. e) Quantification from (c); most KFP-positive Cdk1-null megakaryocytes (n=12) undergo S-G-
    S (endocycles) whereas all (n=14) KFP-positive control megakaryocytes display typical endomitotic cycles (M,
    endomitosis).
    Figure S5. Polyploidization and cell fate of megakaryocytes with Cdk1 and Cdk2-mutant alleles in a
    Cdc20-null background (related to Figure 6). a) Immunodetection of the indicated proteins in total
    megakaryocytes (CD41-positive cells) or high ploidy (≥4C) CD41-positive cells from the bone marrow. -actin
    was used as a loading control. b) Analysis of mitosis (phospho-histone H3; P-H3 signal) in CD41+ bone
    marrow cells from wild-type or Cdc20(); Cdk1(); Cdk2() mice. The ploidy profiles of the whole
    CD41+ population (green), P-H3-positive (red) or P-H3-negative (blue) cells are shown. c) Quantification of
    cells with intermediate levels of ploidy (arrows). Three animals per genotype were analyzed. Data indicate
    mean ± SEM; *, p<0.5; Student’s t-test. d) Cell fate of individual Cdc20(); Cdk1() [left] or Cdc20();
    Cdk1(); Cdk2() [right] bone marrow-derived megakaryocytes recorded by time-lapse microscopy. Cells
    stably expressed histone H2B-GFP, lamin-CFP and geminin-mCherry. Red and green boxes represent S-G2
    (gemininhigh) or G1 (gemininlow) phases, respectively. Endomitosis, prolonged in all cases as a consequence of
    lack of Cdc20, is illustrated as a black box surrounded by a purple frame and red circles represent cell death.
    Supplemental Experimental Procedures

    Mouse colony and histological analysis


    For genotyping the different alleles from the mouse models tail DNA was isolated from 3-4-week old
    mice and PCR amplification reactions were performed using the oligonucleotides described in the
    original manuscripts (Dieguez-Hurtado et al., 2011; Fernandez-Miranda et al., 2011; Manchado et al.,
    2010; Ortega et al., 2003; Tiedt et al., 2007) Cdk1 mutant mice were genotyped using
    oligonucleotides Fw: 5’-GAGATGTAGGATGACTCAGTG-3’ and Rv: 5’-
    TAGCTTATCTACCTCAGCCTG-3’, which generate amplification bands of 650 bp (wild-type allele) or
    800 bp (mutant allele). The following amplification conditions were used: 94ºC during 4 minutes
    followed by 35 cycles of DNA denaturalization at 94ºC during 30 seconds, primer annealing at 60ºC
    during 30 seconds and polymerase extension at 72ºC during 60 seconds ending with a single
    elongation cycle of 7 minutes at 72ºC. For histology sections, sterna and spleens were fixed
    overnight in 4% formalin. Sterna were then decalcified in Decalcifier I solution (Surgipath) for 2 hours.
    Samples were dehydrated in 70% ethanol and processed by the Histopathology Facility Unit at CNIO
    for 3-5 m longitudinal paraffin sections, hematoxylin and eosin staining, or immunohistochemistry.

    Isolation of bone marrow or fetal liver cells and cytometry


    For bone marrow derived megakaryocytes, the tibia and femur were isolated from 8-12 week old
    mice and bone marrow was flushed out by the addition of ice-cold PBS buffer (PBS, 0.5% BSA, 5mM
    EDTA) through the lumen of the bone. Marrow was mechanically disrupted to achieve single cell
    suspension followed by filtering through a 40m nylon strainer to remove bone debris and
    subsequently subjected to erythrocyte lysis. For red blood cell lysis the bone marrow cell pellet from 1
    mouse was resuspended in 1 ml of ACK lysis buffer (150 mM ammonium chloride, 1 mM potassium
    bicarbonate, 0.1 mM EDTA) and incubated for 1.5 min on ice. The lysis was stopped by the addition
    of 10 ml PBS buffer and cells were centrifuged at 200g for 10 min. Cells were counted and subjected
    either directly to surface antigen staining for flow cytometry analysis or to lineage depletion with the
    Miltenyi MACS (magnetic cell shorting) Hematopoietic Progenitor (Stem) Cell Enrichment Set,
    according to manufacturer’s protocol. For fetal liver derived megakaryocytes, cells were obtained
    from whole livers recovered from mouse fetuses between embryonic days 12.5 and 13.5. Single-cell
    suspensions were prepared by successive passage through 19-, 22-, and 25-gauge needles.
    To determine mouse bone marrow hematopoietic stem cells (HSC; Lin IL7R c-kit+ Sca+),
    common lymphoid progenitors (CLP; Lin IL7R+ c-kit+ Sca+), common myeloid progenitors (CMP; Lin
    IL7R c-kit+ Sca CD34+, FcRlow), megakaryocyte, erythroid progenitors (MEP; Lin IL7R- c-kit+ Sca
    CD34, FcRlow), granulocyte, macrophage progenitors (GMP; Lin IL7R c-kit+ Sca CD34+,
    FcRhigh), and megakaryocyte progenitors (LinCD41+) cells, freshly obtained BM cells were stained
    with phycoerythrin (PE) anti-CD34 (BD Biosciences), Alexa Fluor 488 anti- IL7Ra, peridinin
    chlorophyll Protein Cyanin 5.5 (PerCP-Cy 5.5 anti–Sca-1, allophycocyanin-H7 (APC-H7) anti-cKit (BD
    Biosciences), phycoerythrin-cyanin 7 (PE-Cy7) anti-FcR (BD Biosciences) and allophycocyanin
    (APC) -labeled lineage cell detection cocktail (BD Biosciences). Flow cytometric analysis was
    performed with a FACS-Canto flow cytometer or a LSRII flow cytometer (BD Biosciences) and
    FlowJo Version 8.8.7 software (TreeStar).

    Virus preparation
    For preparation of viruses, low passage 293T cells were transfected with the vector of interest and
    the three packaging vectors expressing gag, pol and rev proteins necessary for virion production as
    well the envelope protein vsvg (Tiscornia et al., 2006). 48 h post transfection supernatants containing
    viruses were collected and concentrated by centrigufation at 19.400 rpm for 2 hours at 20 ºC. Viral
    pellets were resuspended in 1x HBSS. This viral preparation is of in vitro grade quality. For
    retroviruses, 293T cells were transfected with the vector of interest and the packaging vector PCL-
    Eco. Supernatants were collected and 48 hours post transfection. Packaging vectors are a gift from
    Dr. Verma´s lab. Transduction of primary megakaryocytes and Lin- bone marrow cells were
    performed by adding 10 l of concentrated virus (stock 109 viral particles per mL) per million of cells
    (MOI 10) for 12 h.

    Supplemental References

    Tiscornia, G., Singer, O., and Verma, I.M. (2006). Production and purification of lentiviral vectors. Nat
    Protoc 1, 241-245.

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