Research ArticleCELL BIOLOGY

Controlled ploidy reduction of pluripotent 4n cells generates 2n cells during mouse embryo development

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Science Advances  16 Oct 2019:
Vol. 5, no. 10, eaax4199
DOI: 10.1126/sciadv.aax4199
  • Fig. 1 Reprogrammed 4n cells contribute to chimeric mice and undergo ploidy reduction in vivo.

    (A) Schematic representation of cell fusion between ESCs and NPCs and subsequent injection of 4n reprogrammed cells (black background) stably expressing pCAG-H2B-tdiRFP or PB-dsRED in 1.5 dpc, two-cell stage embryos (albino background). Representative cell cycle analysis is shown for ESCs, NPCs, and reprogrammed cells. PI, propidium iodide. (B) Representative time-lapse images of an embryo injected with one single PB-dsRED 4n cell. Time is shown in hours:minutes (hh:m). See also movies S1 and S2. Scale bar, 40 μm. DIC, differential interference contrast. (C) Representative immunofluorescence staining of a 5.5 dpc in vitro embryo where a single 4n PB-dsRED cells was injected at 1.5 dpc. Costaining with Nanog (epiblast) and Sox17 (primitive endoderm) markers is shown. Scale bar, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Summary table of chimeric animals obtained after 4n cell injection in 2n blastocysts. Chimerism was assessed on the basis of the presence of tdiRFP-expressing cells by FACS in 12.5 dpc and by coat color and PB-dsRED expression in live-born animals. The asterisks (*) indicate that not enough cells were obtained to perform cell cycle analysis in all chimeric animals. (E) Picture of a representative 8-week-old chimera generated by 4n cell injection in a 2n blastocyst. (F) Bright-field and dsRED fluorescence images of the organs analyzed from the chimeric mouse depicted in (E). (G) FACS profiles of PB-dsRED expression in representative nonchimeric (left) and chimeric (right) organs [chimeric organs are from the chimera depicted in (E)]. PE, phycoerythrin. (H) Cell cycle analysis of sorted PB-dsRED–negative and PB-dsRED–positive cells from the heart of a chimeric mouse FACS-sorted as in (G) (Photo credit: João Frade, Center for Genomic Regulation).

  • Fig. 2 In vitro fusion–derived clones contain 4n and 2n cells and are not aneuploid.

    (A) Quantification of single-cell–derived clones with 4n, mixed (4n + 2n), higher than 4n (>4n), and 2n ploidies (N = 76 clones, two independent fusion experiments; mean ± SD). (B) Representative cell cycle profiles of single-cell–derived clones. (C) Bright-field (top) and Oct4-GFP (middle) images of stable 4n and mixed clones; representative FACS analysis of Oct4-GFP expression is shown on the bottom. FITC, fluorescein isothiocyanate. (D) Top: Cell cycle profile (left) and Oct4-GFP FACS plot (right) of a representative mixed clone (sorting gates are shown in black and green for negative and positive cells, respectively). Bottom: Cell cycle profiles of sorted GFP-negative and GFP-positive cells from a mixed clone. (E) Quantification of karyotypes from cells of stable 4n and mixed clones. Graphs are representative of two stable 4n and four mixed clones (n = number of counted metaphase spreads per clone). Representative metaphase spreads are shown on the right. Scale bars, 20 μm.

  • Fig. 3 4n hybrids undergo bipolar and tripolar mitosis.

    (A) Immunofluorescence images of control ESCs and 4n hybrids in metaphase (DNA, white; β-tubulin, red). Scale bars, 5 μm. (B) Quantification of bipolar and tripolar metaphase spindles in control ESCs, stable 4n, and mixed clones. (ESC: N = 121 cells, two replicates; stable 4n clones: N = 358 cells, two different clones; mixed clones: N = 377 cells, four different clones; mean ± SD). Exact P value is also indicated. (C) Representative time-lapse images of tetraploid cells from mixed clones undergoing bipolar (top) and tripolar (bottom) mitosis (time progression in minutes is shown on each frame). Scale bars, 10 μm. See also movies S12 and S13 (including bright-field images). Schematic representations of the mitotic outcomes are shown on the right side of each panel. (D to F) Quantification of the centrosome number in bipolar metaphases of stable (D) and mixed clones (E) and of tripolar metaphases in mixed clones (F) (mean ± SD). The number of quantified metaphases (n) is indicated in each graph.

  • Fig. 4 Parental chromosome segregation can be nonrandom during tripolar mitosis and generates 2n cells with only one parental genome.

    (A) Quantification of the hybrids’ fate after cell fusion (N = 201 cells, six independent fusion experiments; mean ± SD). (B and C) Representative time-lapse (left) and poststaining images (right) of tripolar mitosis with random (B) or nonrandom (C) chromosome segregation. Merged images correspond to the last frame of the respective time-lapse (time progression in minutes is shown on each frame). Yellow arrowheads indicate EdU-positive cells (pseudocolored magenta) after staining. Scale bars, 10 μm. (D) Schematic representation of two genomic loci carrying different single-nucleotide polymorphisms (SNPs) in ESC (green) and NPC (red). After ploidy reduction, 2n cells can carry one allele from each parental cell or both alleles from only one parental cell. 2n cells derived from nonrandom chromosome segregation during tripolar mitosis include informative SNPs identical to the ones carried by the parental cells. (E) Cell cycle profile of a representative mixed clone from which G1-2n cells (red box) were individually sorted for single-cell SNP genotyping. WGA, whole genome amplification. (F) Percentage of single G1-2n cells that exclusively contain NPC SNPs, ESC SNPs, or mixed SNPs (black, white, and gray bars, respectively) (N = 21 single cells sorted from four different mixed clones; see fig. S4K for SNP percentages of individual cells).

  • Fig. 5 Model of random and nonrandom chromosome segregation during tripolar mitosis of the hybrid cells.

    NPC (red nucleus) and ESC (green nucleus), each carrying a diploid (2n) DNA content, fuse and, after bipolar divisions with centrosome clustering, form tetraploid (4n) synkaryon cells. The generated 4n cells contain four centrosomes and two chromosome sets before mitosis. In the case of tripolar spindle formation, centrosomes do not cluster. If the two parental chromosome sets are spatially separated after nuclear envelope breakdown and are differentially connected to the poles during mitosis, then the parental chromosome segregation will be nonrandom. This results in one 4n cell and two 2n cells that have the same genome as the parental NPC and ESC. Both 2n cells are not aneuploid (a). Alternatively, if parental chromosomes connect randomly to the poles, then the segregation to the three daughter cells is random, resulting in daughter cells with chromosomes from both fusion partners (b).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax4199/DC1

    Fig. S1. Tetraploid Oct4-GFP–positive cells reduce ploidy during embryo development.

    Fig. S2. Fusion-derived cells proliferate in self-renewal conditions and originate Oct4-GFP–positive 4n and Oct4-GFP–negative 2n cells.

    Fig. S3. Tetraploid cells undergo tripolar mitosis and form viable daughter cells.

    Fig. S4. Parental chromosome segregation during hybrids’ division can be nonrandom.

    Fig. S5. 4n-derived 2n cells show NPC and ESC phenotype if cultured in the respective culture medium.

    Movie S1. Time-lapse imaging of an embryo injected with one single PB-dsRED 4n cell. Example 1.

    Movie S2. Time-lapse imaging of an embryo injected with one single PB-dsRED 4n cell. Example 2.

    Movie S3. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 1.

    Movie S4. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 2.

    Movie S5. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 3.

    Movie S6. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 4.

    Movie S7. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 5.

    Movie S8. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 6.

    Movie S9. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 7.

    Movie S10. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 8.

    Movie S11. Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 9.

    Movie S12. Time-lapse images of a synkaryon 4n cell carrying mRFP-tagged histone H2B (H2B-RFP) undergoing bipolar division.

    Movie S13. Time-lapse images of a synkaryon 4n cell carrying H2B-RFP undergoing tripolar division.

    Movie S14. Time-lapse images of a synkaryon 4n cell carrying H2B-RFP undergoing tripolar division without mitotic catastrophe.

    Movie S15. Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP that does not undergo mitosis.

    Movie S16. Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP that undergoes bipolar mitosis.

    Movie S17. Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP that undergoes tripolar mitosis with random segregation.

    Movie S18. Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP that undergoes tripolar mitosis with non-random segregation.

    Movie S19. Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP with parental chromosomes showing different spatial occupancy after bipolar mitosis.

    Movie S20. Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP with parental chromosomes showing different spatial occupancy after tripolar mitosis.

    Movie S21. Time-lapse images of a long-time tracking a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP.

    Table S1. SNP genotyping raw data.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Tetraploid Oct4-GFP–positive cells reduce ploidy during embryo development.
    • Fig. S2. Fusion-derived cells proliferate in self-renewal conditions and originate Oct4-GFP–positive 4n and Oct4-GFP–negative 2n cells.
    • Fig. S3. Tetraploid cells undergo tripolar mitosis and form viable daughter cells.
    • Fig. S4. Parental chromosome segregation during hybrids’ division can be nonrandom.
    • Fig. S5. 4n-derived 2n cells show NPC and ESC phenotype if cultured in the respective culture medium.
    • Legends for movies S1 to S21
    • Legend for table S1

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Time-lapse imaging of an embryo injected with one single PB-dsRED 4n cell. Example 1.
    • Movie S2 (.avi format). Time-lapse imaging of an embryo injected with one single PB-dsRED 4n cell. Example 2.
    • Movie S3 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 1.
    • Movie S4 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 2.
    • Movie S5 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 3.
    • Movie S6 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 4.
    • Movie S7 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 5.
    • Movie S8 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 6.
    • Movie S9 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 7.
    • Movie S10 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 8.
    • Movie S11 (.avi format). Fly-through images of CT studies performed in 8-week-old chimeric mouse. Mouse 9.
    • Movie S12 (.avi format). Time-lapse images of a synkaryon 4n cell carrying mRFP-tagged histone H2B (H2B-RFP) undergoing bipolar division.
    • Movie S13 (.avi format). Time-lapse images of a synkaryon 4n cell carrying H2B-RFP undergoing tripolar division.
    • Movie S14 (.avi format). Time-lapse images of a synkaryon 4n cell carrying H2B-RFP undergoing tripolar division without mitotic catastrophe.
    • Movie S15 (.avi format). Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP that does not undergo mitosis.
    • Movie S16 (.avi format). Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP that undergoes bipolar mitosis.
    • Movie S17 (.avi format). Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP that undergoes tripolar mitosis with random segregation.
    • Movie S18 (.avi format). Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP that undergoes tripolar mitosis with non-random segregation.
    • Movie S19 (.avi format). Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP with parental chromosomes showing different spatial occupancy after bipolar mitosis.
    • Movie S20 (.avi format). Time-lapse images of a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP with parental chromosomes showing different spatial occupancy after tripolar mitosis.
    • Movie S21 (.avi format). Time-lapse images of a long-time tracking a sorted hybrid cell generated after fusion between ESC-H2B-eGFP and NPC-H2B-mRFP.
    • Table S1 (Microsoft Excel format). SNP genotyping raw data.

    Files in this Data Supplement:

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