Laboratory of Chromosome Dynamics and Evolution

National Heart, Lung, And Blood Institute

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Abstract

Mechanisms of non-Mendelian segregation in female meiosis To understand how meiotic drive elements achieve non-Mendelian segregation, we currently focus on selfish R2d2, a non-centromeric locus on mouse chromosome 2. This selfish element shows over 90% transmission ratio distortion with mild embryonic lethality. The underlying cell biological basis for both biased segregation in female meiosis and the embryonic lethality is unknown. To study the dynamics of the R2d2 locus during female meiosis, we developed two strategies to visualize this locus in mouse oocytes. First system is based on the recently developed FISH technique called Oligo-paint. In collaboration with Dr. Leah Rosin (NICHD), we implemented the Oligo-paint technique to mouse oocyte cells. Oligo-paint of the R2d2 locus would allow us to test if the R2d2 locus has different chromatin structure compared to the neighboring regions and perform IF-FISH to screen what proteins are recruited to the R2d2 locus to cheat the segregation process. We have analyzed the recombination landscape around the R2d2 locus using this Oligo-paint technique and found that selfish R2d2 is cheating more often in meiosis II division than in meiosis I. To understand how selfish R2d2 is cheating in meiosis II, we developed another method to live-image this locus based on the CRISPR/Cas9 technique. In this method, we microinject mouse oocytes with a deactivated version of Cas9 protein fused to GFP (dCas9-GFP) in complex with sgRNA that recognize the R2d2 DNA sequence. dCas9-GFP specifically localized to the R2d2 locus, which allowed us to perform live-cell imaging to capture the cheating behaviors in action. The live-imaging reveled that selfish R2d2 cheats in a different way from selfish centromeres, which bias their orientation towards the egg side on the spindle in metaphase I. Meiotic drive is fundamental to sexual reproduction and has been recognized as a powerful force in genetics and evolutionary biology since first described in maize in 1942. The underlying mechanisms have long been mysterious to cell biologists. This project tackles this exciting problem, all the way from developing experimental systems to revealing how selfish elements challenge Mendel and affect fitness. Moreover, our work will lead to a deeper understanding of the interactions between chromosomes and spindle microtubules. These interactions are highly error-prone in humans and a major cause of infertility, which could be caused by selfish behaviors of meiotic drive elements. Mechanisms of reproductive isolation through hybrid female sterility Reproductive isolation occurs when the genomes of two populations accumulate genetic incompatibilities that prevent inter-breeding. For example, hybrid incompatibility in the meiotic chromosome segregation process would lead to the formation of aneuploid gametes and fertility defects. Indeed, there are multiple studies, showing hybrid animals having reduced fertility, serving as a reproductive isolating barrier. However, the molecular basis causing reduced hybrid fertility is largely unknown especially in mammals. We currently study hybrid fertility issues using an interspecific mouse hybrid between Mus musculus and Mus spretus, because the hybrid mice are viable but have fertility defects in both sexes. Hybrid females are known to produce aneuploid eggs, implying hybrid incompatibility in the meiotic chromosome segregation process. We combine high-resolution light microscopy with experimental manipulation of chromosome dynamics to reveal the molecular basis underlying this meiotic failure. Elucidating the mechanisms of hybrid fertility defects will lead to a deeper understanding of the speciation process. To understand why M. musculus x M. spretus hybrid oocytes produce aneuploid eggs, we examined the chromosome structure in meiosis I oocytes. We found that hybrid oocytes have meiotic bivalents with M. musculus centromeres extremely stretched, while M. spretus centromeres remained condensed. Centromere stretching caused mis-attachment with spindle microtubules, leading to lagging chromosomes in anaphase. Such segregation defects explain why this hybrid female produces aneuploid eggs and show fertility defects. However, there were two big questions remaining: (1) why there is chromosome condensation defects in this hybrid, and (2) why it is specific to M. musculus centromeres. In mouse oocytes, condensin II complex is the major condensin responsible to form the rod-shape chromosome structure. Therefore, we focused on condensin II and found that hybrid oocytes have significantly less condensin II on their chromosomes. This observation indicates that the condensin II loading is less efficient in the hybrid genetic background. By carefully analyzing the condensin II localization within the chromosome, we found that condensin II was especially low at the M. musculus peri-centromere region, composed of Major satellite repeat sequences. Consistently, it was the Major satellite region that was stretching on the M. musculus centromere in hybrid oocytes. In contrast to the M. musculus genome, where Major satellite is the most abundant satellite, comprising 10% of the genome, the M. spretus genome has very little Major satellite repeats. Therefore, M. spretus centromeres load condensin II well and remain condensed even in the hybrid genetic background where condensin II amount is overall reduced on the chromosome. Altogether, this asymmetric condensin II localization can explain the species-specific condensation failures at centromeres, leading to the production of aneuploid eggs in this hybrid system. Overall, this project demonstrated that the chromosome condensation failure can serve as a reproductive isolating barrier in mice. Multiple condensin subunit genes are under rapid evolution, leading to significant sequence divergence even between closely related species. Therefore, condensin genes are especially attractive candidates causing hybrid incompatibility. Since condensin subunits are rapidly evolving in multiple taxa, including Drosophila and several mammalian clades, condensin mis-regulation may be creating reproductive isolating barriers in other lineages as well.