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Dual-spindle formation in zygotes keeps parental genomes apart in early mammalian embryos

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Science  13 Jul 2018:
Vol. 361, Issue 6398, pp. 189-193
DOI: 10.1126/science.aar7462
  • Fig. 1 Individual bipolar spindle formation around each pronucleus.

    (A) Time-lapse imaging of Mus musculus x Mus musculus (MMU x MMU) zygotes expressing EB3-mCherry (marker for microtubules; green) and tdEos-Cep192 (marker for MTOCs; magenta). Scale bar, 10 μm. In 10 out of 13 zygotes, both or at least one of the dual-spindle poles remained clearly split after the spindles had parallelized. (B) Schematic diagram showing progression of dual-spindle formation based on the data presented in (A) and data shown subsequently in the manuscript. Microtubules, gray; MTOCs, magenta. (C) Immunofluorescence staining of MMU x MMU zygotes fixed at consecutive stages of development. Maximum z projections of confocal sections of zygotes at prophase, early pro-metaphase, late pro-metaphase, and early metaphase. White arrowheads indicate poles. (D) Immunofluorescence staining of cold-treated MMU x MMU pro-metaphase zygotes. Maximum z projections of confocal sections. Microtubules, α-Tubulin (green); MTOCs, Pericentrin (magenta); kinetochores, Crest (white); and DNA, Hoechst (blue). Scale bars, 5 μm in (C) and (D).

  • Fig. 2 Spindle assembly and chromosome dynamics in the zygote.

    (A) Time-lapse imaging of Mus musculus x Mus Spretus (MMU x MSP) zygotes expressing EB3-mCherry and fluorescent TALEs to label maternal and paternal chromosomes though distinction of Major satellites (MajSat) and Minor satellites (MinSat). Phase 1: Microtubule ball formation around pronuclei. Phase 2: Bipolarization of maternal and paternal spindle. Phase 3: Formation of single barrel–shaped spindle. (Top and top middle) 3D-rotated images of the whole spindle volume. Maternal chromosomes, MajSat (magenta); paternal chromosomes, MinSat (cyan); microtubules, EB3-mCherry (white). (Bottom middle and bottom) Segmentation of maternal (MatSpd; magenta) and paternal (PatSpd; cyan) spindles in phase 1 and phase 2 and single bipolar spindle in phase 3 (CompoundSpd; gray). Offset between maternal and paternal chromosomes at metaphase and anaphase is indicated with white arrows. (B) Schematic of measurements on maternal (MatChr) and paternal chromatin masses (PatChr) in phases 2 and 3. (C and D) Angle between maternal and paternal chromosome axis over time for (C) a single embryo and (D) averaged for 12 embryos (mean ± SD) is shown. Phase 1, blue; phase 2, red; phase 3, green.

  • Fig. 3 Proximity dependency of bipolar spindle fusion.

    (A) Time-lapse imaging of MMU x MMU zygotes expressing H2B-mCherry (chromatin; magenta) and αTubulin–enhanced green fluorescent protein (EGFP) (microtubules; green). Shown is spindle morphology from pro-metaphase to postmitosis in three representative zygotes treated with Nocodazole for >10 hours. Maximum z projections are of pro-metaphase, metaphase, anaphase, telophase, and postmitosis. Arrowheads and “PB” indicate nuclei and polar body, respectively. Scale bar, 10 μm. In the absence of NEBD as a timing reference, anaphase onset was set at 90 min (average time from NEBD to anaphase in MMU x MMU zygotes), and the other times were calculated accordingly. (B) Initial distance of pro-nuclei (n = 19). Statistics, Student’s t test.

  • Fig. 4 Distribution of parental centromeres in control, NocMo-treated, and MoNoc-treated zygotes.

    (A) Differential labeling of maternal (MajSat; magenta) and paternal (MinSat; cyan) centromeres through distinction of single-nucleotide polymorphisms by means of fluorescent TALEs. Mitotic spindle is labeled with EB3-mCherry (white). Representative z-projected images of parental chromosome distribution in untreated, MoNoc, and NocMo MMU x MSP zygotes. Scale bar, 10 μm. (B) Degree of overlap between 3D convex hulls of parental chromosomes for untreated (n = 31), MoNoc (n = 16), and NocMo (n = 12) zygotes and embryos with in silico randomized distribution (n = 40) (fig. S1 and supplementary materials, materials and methods). Statistics, Student’s t test.

Supplementary Materials

  • Dual-spindle formation in zygotes keeps parental genomes apart in early mammalian embryos

    Judith Reichmann, Bianca Nijmeijer, M. Julius Hossain, Manuel Eguren, Isabell Schneider, Antonio Z. Politi, Maria J. Roberti, Lars Hufnagel, Takashi Hiiragi, Jan Ellenberg

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S10
    • Table S1
    • References

    Images, Video, and Other Media

    Movie S1
    Live-cell time-lapse imaging of MMU x MSP mouse zygote expressing fluorescent TALEs for differential labelling of maternal (magenta) and paternal (cyan) centromeres through distinction of minor and major satellite regions. Chromosome arms are labelled with H2B-mCherry (grey). Time resolution is 7.5 min.
    Movie S2
    Live-cell time-lapse imaging of MMU mouse zygote expressing EB3-mCherry (green) and tdEos-Cep192 (magenta). Time in min.
    Movie S3
    Movie S4
    Movie S5
    Movies S3-5. Live-cell time-lapse imaging of pro-metaphase mouse zygote (phase 2) expressing EGFP-EB3 Left panel: Maximum intensity Z projection. Right panel: The movie has been filtered with a Laplacian of Gaussian (2x2 pixels) to enhance the EB3 tip signal (right panel). Scale bar 10 μm. Time resolution is 800 ms. Movies S3-5 represent the three examples corresponding to Figure S5A.
    Movie S6
    Movie S7
    Movie S8
    Movies S6-8.
    Live-cell time-lapse imaging of metaphase mouse zygote (phase 3) expressing EGFPEB3 Left panel: Maximum intensity Z projection. Right panel: The movie has been filtered with a Laplacian of Gaussian (2x2 pixels) to enhance the EB3 tip signal (right panel). Scale bar 10 μm. Time resolution is 800 ms.
    Movies S6-8 represent the three examples corresponding to Figure S5B.
    Movie S9
    Movie S10
    Movie S11
    Movies S9-11.
    Live-cell time-lapse imaging of MMU mouse zygote after washout of Nocodazole (> 10 h treatment) expressing αTubulin-EGFP (green) and H2B-mCherry (magenta). Time in min. Movies S9-11 represent three examples corresponding to Figure 3.

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