Micronucleus formation causes perpetual unilateral chromosome inheritance in mouse embryos

Abstract

Chromosome segregation defects in cancer cells lead to encapsulation of chromosomes in micronuclei (MN), small nucleus-like structures within which dangerous DNA rearrangements termed chromothripsis can occur. Here we uncover a strikingly different consequence of MN formation in preimplantation development. We find that chromosomes from within MN become damaged and fail to support a functional kinetochore. MN are therefore not segregated, but are instead inherited by one of the two daughter cells. We find that the same MN can be inherited several times without rejoining the principal nucleus and without altering the kinetics of cell divisions. MN motion is passive, resulting in an even distribution of MN across the first two cell lineages. We propose that perpetual unilateral MN inheritance constitutes an unexpected mode of chromosome missegregation, which could contribute to the high frequency of aneuploid cells in mammalian embryos, but simultaneously may serve to insulate the early embryonic genome from chromothripsis.A ccurate chromosome segregation is achieved by correct attachment of spindle microtubules to kinetochores, complex proteinaceous structures that assemble on centromeric DNA. Misattachment can cause so-called lagging chromosomes during anaphase (1), which are a hallmark of chromosomally unstable cells (2), and can result in micronuclei (MN)-small nucleus-like bodies that form if a chromosome remains separate from the main group of chromosomes at the time of nuclear envelope reformation. MN have long been used as a marker of genetic fidelity. For example, MN may be predictive of tumorigenicity and can be used to screen chemicals for genotoxicity (3-5). However, the cellular impact of MN formation is poorly understood. Recent studies found that chromosomes within MN become heavily damaged, causing DNA rearrangements (6-9). Paired with reincorporation of the MN chromosome into the main nucleus (principal nucleus; PN) during the next cell cycle (7, 9, 10), this provided an elegant explanation for chromosome-specific extensive rearrangements seen in many cancer cells, termed chromothripsis (11,12). In the present study we show that the outcome of MN formation is markedly different in the preimplantation mammalian embryo.Chromosomal mosaisicm is common in mammalian preimplantation embryos, up to 50% of human embryos produced in fertility clinics containing some aneuploid cells resulting from early mitotic errors (13-16), for which there is currently no clear cellular explanation. Simultaneously, early embryos frequently exhibit MN, the causes and consequences of which are unknown (17, 18). Here we pair long-term live 4D microscopy with high-resolution fixed embryo analysis to analyze the causes and consequences of naturally occurring MN in mouse embryos. Our experiments reveal an unexpected series of events in which chromosomes from within MN do not rejoin the principal nucleus, but are repeatedly inherited by only one daughter cell. We propose that this mechanism should generate a cascade of aneuploi...

Causes and consequences of chromosome segregation error in preimplantation embryos

“…As described earlier, it appears increasingly plausible that mosaic aneuploidy is, perhaps counter intuitively, a normal feature of preimplantation development across multiple mammalian species (Dupont et al 2010, Hornak et al 2012, Mizutani et al 2012, Bolton et al 2016, Vázquez-Diez et al 2016. In which case, it becomes reasonable to ask; could chromosome missegregation in the early embryo present some positive biological benefit?…”

“…4), the micronucleus is unilaterally inherited at each subsequent cell division (Vázquez-Diez et al 2016). This can explain two of the most commonly observed features of mosaicism in the clinic.…”

“…Additionally, PLK4 has been shown to be essential for bipolar spindle assembly in mouse preimplantation embryos, despite the fact that centrioles are not present, further indicating non-canonical roles of well-characterised spindle proteins in early development (Coelho et al 2013, Arquint & Nigg 2016. Although centrioles later emerge at blastocyst stage, it remains unclear whether they are fully functional, since they appear then to lack (Fragouli et al 2013, Vázquez-Diez et al 2016. By extensive imaging of embryonic mitoses in mouse it was shown that micronuclei arise because of single sister chromatids that remain separate from the main group in anaphase (termed 'lagging chromosomes'), which then form their own nuclear membrane in the resulting daughter cells (Fig.…”

“…4 and Video 1,). Following encapsulation into micronuclei, the chromosomes become extensively damaged and lose centromeric identity indicated by the centromere marker CENP-A, and therefore, fail to assemble a kinetochore and be segregated by the spindle during subsequent cells divisions (Vázquez-Diez et al 2016). A similar series of events has been observed in somatic cells (Guerrero et al 2010, Crasta et al 2012, Huang et al 2012, Zhang et al 2015a, and it is thought that the defective nuclear PROOF ONLY envelope on the micronucleus, leading to failed nuclear compartmentalisation, underpins the DNA damage to the chromosome enclosed (Hatch et al 2013).…”

“…A similar series of events has been observed in somatic cells (Guerrero et al 2010, Crasta et al 2012, Huang et al 2012, Zhang et al 2015a, and it is thought that the defective nuclear PROOF ONLY envelope on the micronucleus, leading to failed nuclear compartmentalisation, underpins the DNA damage to the chromosome enclosed (Hatch et al 2013). Strikingly, in embryos, the same heavily damaged chromosome re-appears as a micronucleus following each subsequent cell division (Vázquez-Diez et al 2016). Although live imaging of this type in human embryos is yet to be reported, human embryo immunofluorescence data show the occurrence of single anaphase lagging chromosomes, micronuclei bearing high levels of DNA damage and lacking centromeric identity, and micronucleus-like chromosomes in mitosis devoid of kinetochore staining (Kort et al 2016).…”