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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

It takes two to tango

Fusion of egg and sperm combines the genetic material of both parents in one cell. In mammals, including humans, each parental genome is initially confined in a separate pronucleus. For the new organism to develop, the two genomes must be spatially coordinated so that the first embryonic division can create two cells that combine both genomes in one nucleus. Reichmann et al. found that at the beginning of the first division, two microtubule spindles organize the maternal and paternal chromosomes and subsequently align to segregate the parental genomes in parallel (see the Perspective by Zielinska and Schuh). Failure of spindle alignment led to two-celled embryos with more than one nucleus per cell. Dual-spindle assembly in the zygote thus offers a potential mechanistic explanation for division errors frequently observed in human embryos in the fertility clinic.

Science, this issue p. 189; see also p. 128

Abstract

At the beginning of mammalian life, the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible for spatially combining the two genomes and then segregating them to create the two-cell embryo. We used light-sheet microscopy to show that two bipolar spindles form in the zygote and then independently congress the maternal and paternal genomes. These two spindles aligned their poles before anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a potential rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.

After fertilization, the haploid genomes of egg and sperm come together to form the genome of a new diploid organism, a moment that is of fundamental biological importance. In mammals, parental chromosomes meet for the first time upon entry into the first zygotic mitosis after nuclear envelope breakdown (NEBD). It has been assumed that, as observed in oocytes, a single bipolar microtubule system would self-assemble around both parental genomes in zygotes (16). However, owing to the extreme light sensitivity of the mammalian embryo, the details of the dynamic process of zygotic spindle assembly remain unclear.

To examine how parental genomes join for the first time, we imaged live embryos in which the maternal and paternal centromeres were differentially labeled (7) using our recently developed inverted light-sheet microscope, which allows fast three-dimensional (3D) imaging of embryonic development owing to its low phototoxicity (8). This revealed that the two genomes remain spatially separate throughout the first mitosis (fig. S1 and movie S1). To understand why the genomes are not mixed, we next imaged spindle assembly using fluorescently labeled microtubule organizing centers (MTOCs) and spindle microtubules (Fig. 1A and movie S2). Newly nucleated microtubules self-organized into two separate bipolar spindles after NEBD, attracting a subset of the cytoplasmic MTOCs that had accumulated around each pronucleus to their poles (Fig. 1, A and B). Subsequently, the two spindles aligned and came into close apposition to form a compound barrel–shaped system. This structure typically had two clusters of MTOCs at at least one of its poles, suggesting that the two spindles were aligned closely but not completely merged (Fig. 1, A and B, and movie S2). To probe this further, we performed 3D immunofluorescence analysis of zygotes, visualizing endogenous spindle poles, microtubules, kinetochores, and DNA. In early and mid pro-metaphase, two separate bipolar spindles were formed in in vivo–developed zygotes (Fig. 1, C and D). Given the delayed and partial association of MTOCs with the microtubule mass, we hypothesized that dual-spindle formation might be driven by self-assembly of microtubules nucleated by chromosomes. To test this, we assayed microtubule regrowth after washing out the microtubule-depolymerizing drug Nocodazole. Indeed, a large proportion of microtubules was nucleated on chromosomes, particularly at kinetochores (fig. S2), whereas MTOCs became associated with microtubules only later. This observation prompted us to investigate the organization of K-fibers in zygotic pro-metaphase by means of high-resolution immunofluorescence of zygotes fixed after brief cold treatment in order to highlight stable microtubules (Fig. 1D and fig. S3). Two bipolar arrays of K-fibers started assembling in early pro-metaphase and were stably organized in mid pro-metaphase, and their remnants were clearly recognizable by their slightly offset centers and split poles even after parallelization in metaphase, during which the two spindles were no longer separated by a large gap.

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).

To characterize the kinetics of zygotic spindle assembly in live embryos, we next imaged maternal and paternal centromeres in relation to growing spindle microtubule tips and observed three phases of zygotic spindle assembly (Fig. 2A). A transient first phase (~3 min; 10.3 ± 3.5 to 13. 4 ± 4 min after NEBD), characterized by the clustering of growing microtubules around the two pronuclei, was followed by phase 2 (~16 min; 14.5 ± 4 to 30.7 ± 6.5 min after NEBD), in which individual bipolar spindles assembled around each parental genome, and subsequently, phase 3 (~83 min; 46.7 ± 17 to 129.2 ± 16.5 min after NEBD), when the two spindles aligned and combined into a compound barrel–shaped structure.

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.

To test whether the two zygotic spindles are functionally independent, we measured the timing and direction of maternal and paternal chromosome congression (fig. S4, A and B, and supplementary materials, materials and methods). Congression started in pro-metaphase (phase 2), while the spindles were clearly separated (fig. S4, A and B). Parental chromosome congression was not correlated in time until shortly before anaphase, suggesting that they are moved by different microtubule systems (fig. S4C). Furthermore, the parental genomes were congressed in different directions along separate spindle axes, as evidenced by the large difference between the angles of the two forming metaphase plates, which became parallel only later during dual spindle alignment in phase 3 (Fig. 2, B to D, and fig. S4, D and E). Tracking of growing microtubule tips showed two major directions of microtubule flow during phase 2 and one main direction during phase 3, as indicated by the corresponding kymograph profiles (fig. S5 and movies S3 to S8). The independent congression frequently led to an offset in the final bioriented position of the paternal and maternal metaphase plates at the end of phase 3 before and during segregation (Fig. 2A, arrows). Thus, each of the two spindles around the parental genomes functions independently for chromosome congression and may even function separately in chromosome segregation.

In in vitro fertilization (IVF) clinics, the zygotic division is error prone and often leads to embryos with blastomeres that contain two nuclei (913). We hypothesized that failure to align the two parental spindles before anaphase could explain this enigmatic phenotype. To test this, we increased the distance between the two pronuclei with transient treatment with Nocodazole, which led to a larger gap between the two self-assembling spindles (Fig. 3 and movies S9 to S11). Indeed, such embryos frequently failed to fully align the parental spindles at one or both poles. This did not delay anaphase but resulted in chromosome segregation by two spindles into different directions, leading to two cell embryos with one or two binucleated blastomeres (Fig. 3 and movies S10 and S11). By contrast, embryos that did align the two spindles parallel to each other before anaphase cleaved into two blastomeres with single nuclei (movie S9). Thus, failure to align the two zygotic spindles gives rise to multinucleated two-cell embryos, phenocopying frequently observed errors in human embryonic development in IVF clinics.

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.

Dual-spindle assembly in the mammalian zygote would also offer a mechanistic explanation for the long-standing observation that the parental genomes occupy separate compartments inside the nuclei of two- and four-cell embryo blastomeres (14, 15). If dual-spindle assembly around two pronuclei was responsible for genome compartmentalization, parental genomes should mix in subsequent divisions, in which only one nucleus is present per cell. Imaging of the metaphase plate of live hybrid mouse embryos from the zygote to the eight-cell stage showed that parental genomes were separated in zygotes but rapidly became mixed in the subsequent developmental stages, as predicted (fig. S6, A to D). This loss of genome compartmentalization was also seen in in vivo–developed isogenic embryos (fig. S6, E to I). Thus, parental genomes are kept separate by two spindles only during the first mitosis but then mix during subsequent divisions, driven by a single common spindle.

If dual-spindle assembly is the mechanism for parental genome compartmentalization (fig. S7A), formation of a single spindle around both genomes in the zygote should already mix them in the first division. To test this prediction, we redirected spindle assembly with two small-molecule inhibitors of microtubule polymerization (Nocodazole) and the motor protein Eg5 (Monastrol). Transient treatment with Monastrol collected both genomes in a single microtubule aster, and subsequent depolymerization of microtubules with Nocodazole followed by regrowth after washout then resulted in one bipolar spindle around both genomes (figs. S7B and S8, MoNoc-treated zygotes). Such MoNoc-treated embryos captured and congressed chromosomes within a single spindle and showed a high degree of parental genome mixing in the first mitotic metaphase (Fig. 4). This was substantially different from untreated or control zygotes in which the order of drug treatments is reversed (NocMo-treated zygotes), which maintained dual-spindle formation and genome separation (Fig. 4 and figs. S7C and S8). Thus, dual-spindle formation in the zygote is required for parental genome separation in mammals.

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.

Having this experimental method to induce mixing of the parental genomes in hand allowed us to demonstrate that genome separation was not required for parental genome epigenetic asymmetry and its resolution (1619), as proposed previously (figs. S9 and S10) (15, 2022).

Here, we showed that two spindles form around pronuclei in mammalian zygotes, which individually collect the parental genomes and then position them next to each other before the first anaphase by aligning the two spindles parallel to each other. Our data explain how parental genome separation is achieved in mammalian embryos. To date, the formation of physically distinct mitotic spindles around the two pronuclei has been thought to be specific to certain arthropod species (23, 24). Our finding that this occurs also during mammalian pro-metaphase suggests that two zygotic spindles might be characteristic for many species that maintain separate pronuclei after fertilization. Failure to align the two spindles produced errors in the zygotic division that closely resemble clinical phenotypes of human embryos in IVF procedures, suggesting that a similar mechanism of dual zygotic spindle assembly also occurs in humans. This view is supported by the spatial separation of parental chromosomes reported in human zygotes (25) and divisions of human zygotes into multinucleated blastomeres, as well as by reports that identified a mix of paternal, maternal, and diploid cells in eight-cell cattle embryos. A dual zygotic spindle would provide a mechanistic basis for this parental genome segregation (913, 26, 27). These severe and relatively frequent zygotic division errors in human and agriculturally used mammals thus find their likely mechanistic explanation in a failure of the close alignment of the two zygotic spindles before anaphase. If a similar mechanism of microtubule-driven parental genome separation indeed occurs in human zygotes, it would be important from an ethical and legal perspective because “pronuclear fusion,” a process that strictly speaking does not occur in mouse zygotes, is used to define the beginning of embryonic life as protected by law in several countries (for example, Germany, § 8 Abs. 1 Embryonenschutzgesetz).

Supplementary Materials

www.sciencemag.org/content/361/6398/189/suppl/DC1

Materials and Methods

Figs. S1 to S10

Table S1

Movies S1 to S11

References (2834)

References and Notes

Acknowledgments: We thank N. Daigle for cloning the EB3-mCherry plasmid; P. Neveu and M. Schuh for providing tdiRFP670 and tdEos-Cep192, respectively; and N. Galjart for providing full-length Homo sapiens EB3 cDNA (NM_001303050.1). We thank EMBL’s laboratory of animal resources for support, P. Strnad for development and assistance of the inverted light-sheet microscope, A. Rybina for assistance with the Zeiss LSM 880 Airy microscope, Arivis for support in image analysis, and the EMBL Advanced Light Microscopy Facility for support in image acquisition and analysis. We thank J. Reddington and S. Alexander for critical reading of the manuscript. Funding: This work was supported by funds from the European Research Council (ERC Advanced Grant “Corema,” grant agreement 694236) to J.E. and by the European Molecular Biology Laboratory (all authors). J.R. was further supported by the EMBL Interdisciplinary Postdoc Programme (EIPOD) under Marie Curie Actions COFUND; M.E. by the EMBO long-term postdoctoral fellowship and EC Marie Slodowska-Curie postdoctoral fellowship; I.S. by a Boehringer Ingelheim Fonds Ph.D. fellowship; and M.J.R. by a Humboldt Foundation postdoctoral fellowship. I.S. is a candidate for a joint Ph.D. between EMBL and Heidelberg University, Faculty of Biosciences. Author contributions: J.E. and J.R. conceived the project and designed the experiments. J.R., B.N., M.E., and I.S. performed the experiments. M.J.R. supported the mouse EDU experiments. J.R., M.J.H., and A.Z.P. analyzed the data. T.H. and L.H. contributed to conception and design of the work. J.E. and J.R. wrote the manuscript. All authors contributed to the interpretation of the data and read and approved the final manuscript. Competing interests: L.H. and J.E. are scientific cofounders and advisers of Luxendo GmbH (part of Bruker), which makes light-sheet–based microscopes commercially available. Data and materials availability: All images processed in this study are available in the Image Data Resource (IDR), accession number idr0045 (https://idr.openmicroscopy.org/webclient/?show=project-405). All code is available from EMBL’s git depository (https://git.embl.de/grpellenberg/dual_zygotic_spindles_reichmann_2018).
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