Periodic Reporting for period 4 - COREMA (Cell division and the origin of embryonic aneuploidy in preimplantation mouse development)
Okres sprawozdawczy: 2021-07-01 do 2021-12-31
Cell division is fundamental for the early development of the mammalian embryo. It drives the rapid early proliferation of totipotent cells and formation of a multicellular organism. Strikingly, the reported incidence of chromosome aberrations in early human embryos exceeds 50% and hence is a major cause of infertility and congenital diseases. Why aneuploidy (presence of an abnormal number of chromosomes in a cell) is so prevalent at the beginning of mammalian life and how development nevertheless achieves robustness is fundamentally not understood. The overall goal of the proposed research is to characterise the cell divisions of the mouse embryo occurring before the embryo implants into the uterus of its mother in order to mechanistically understand which errors in cell division processes cause aneuploidy and what the consequences for early development are. Enabled by new imaging technology development for live embryo imaging, we will map when, how and why cell division goes awry, identify the key mitotic pathways that underlie aneuploidy generation, and dissect the responsible molecular mechanism(s).
The proposed work combined an interdisciplinary approach of parallel technology development and biological, functional and molecular studies.
(1) Technology development:
To enable imaging of the long-term fate of individual chromosomes over several divisions in the living preimplantation mouse embryo, we designed the 2nd generation inverted light sheet microscope (iSPIM2) which features extension of a thin light sheet through the entire embryo volume while decreasing the applied light dose to be able to image all blastomeres with high resolution and low phototoxicity. The final design offers thin light sheets with tunable properties and dual, high-powered detection objectives result in high resolution, high contrast views throughout the embryo volume. The iSPIM2 has been validated for multiple samples including mouse embryos (Kromm, Lin, et al., in preparation; patent application has been filed). In parallel we developed computational tracking pipelines to construct a 4D chromosome map of mouse preimplantation development. An open-source tracking implementation is publicly available (https://git.embl.de/grp-ellenberg/kinetochore_tracking).
(1) Biological studies:
The first embryonic division is of particular importance as it facilitates the union of the maternal and paternal genomes. In the past it has been believed that a single spindle combines the maternal and paternal chromosomes. However, we could show that two bipolar spindles form in the zygote which independently congress the maternal and paternal chromosomes, keeping the parental genomes apart during the first cleavage. This intriguing spindle assembly mechanism provides a potential rationale for the 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 (Reichmann et al., doi:10.1126/science.aar7462). While the zygotic division faces the special challenge of organising two initially separate parental genomes in two spindles, also the subsequent embryonic divisions are highly error prone. We therefore extended our study to characterize the first five cell divisions. To unravel molecular mechanisms of embryonic aneuploidy generation we tested the different mechanisms potentially involved in aneuploidy generation and it became clear that likely an interplay and synergy between several mechanisms is responsible. We also investigated spindle assembly in the bovine system, where (as in humans) only two MTOCs (centrosomes) are present after fertilisation and centriole duplication. In collaboration with Tom Stout and Marta de Ruijter-Villani from the Department of Clinical Sciences, Faculty of Veterinary Medicine at Utrecht University, we found that the two spindles also frequently form even in the presence of the two centrosomes. We showed that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We concluded that the dual spindle assembly pathway is conserved in nonrodent mammals, which could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos (Schneider et al., doi:10.1083/jcb.202010106).
The discovery of the existence of the dual spindle resulted in a broad media coverage across the world and interest by the general public. The impact of this work was highlighted in several German newspapers and magazines (FAZ, Zeit, Süddeutsche, Spektrum der Wissenschaft and EMMA) as well as by a Science perspective article written by Melina Schuh, a Developmental Cell preview article by Michael A. Lampson and a Dispatch in Current Biology article by Marie-Hélène Verlhac. The relevance of our findings for reproductive medicine is furthermore reflected by its influence on ethical discussions especially in Germany. In this context Reichmann et al. was cited in the Leopoldina (German Academy of Science) recommendation for a new law for reproductive medicine presented to the German government in 2021 (https://www.leopoldina.org/uploads/tx_leopublication/2021_Stellungnahme_Embryonenschutz_web.pdf).
(1) Technology development:
To enable imaging of the long-term fate of individual chromosomes over several divisions in the living preimplantation mouse embryo, we designed the 2nd generation inverted light sheet microscope (iSPIM2) which features extension of a thin light sheet through the entire embryo volume while decreasing the applied light dose to be able to image all blastomeres with high resolution and low phototoxicity. The final design offers thin light sheets with tunable properties and dual, high-powered detection objectives result in high resolution, high contrast views throughout the embryo volume. The iSPIM2 has been validated for multiple samples including mouse embryos (Kromm, Lin, et al., in preparation; patent application has been filed). In parallel we developed computational tracking pipelines to construct a 4D chromosome map of mouse preimplantation development. An open-source tracking implementation is publicly available (https://git.embl.de/grp-ellenberg/kinetochore_tracking).
(1) Biological studies:
The first embryonic division is of particular importance as it facilitates the union of the maternal and paternal genomes. In the past it has been believed that a single spindle combines the maternal and paternal chromosomes. However, we could show that two bipolar spindles form in the zygote which independently congress the maternal and paternal chromosomes, keeping the parental genomes apart during the first cleavage. This intriguing spindle assembly mechanism provides a potential rationale for the 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 (Reichmann et al., doi:10.1126/science.aar7462). While the zygotic division faces the special challenge of organising two initially separate parental genomes in two spindles, also the subsequent embryonic divisions are highly error prone. We therefore extended our study to characterize the first five cell divisions. To unravel molecular mechanisms of embryonic aneuploidy generation we tested the different mechanisms potentially involved in aneuploidy generation and it became clear that likely an interplay and synergy between several mechanisms is responsible. We also investigated spindle assembly in the bovine system, where (as in humans) only two MTOCs (centrosomes) are present after fertilisation and centriole duplication. In collaboration with Tom Stout and Marta de Ruijter-Villani from the Department of Clinical Sciences, Faculty of Veterinary Medicine at Utrecht University, we found that the two spindles also frequently form even in the presence of the two centrosomes. We showed that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We concluded that the dual spindle assembly pathway is conserved in nonrodent mammals, which could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos (Schneider et al., doi:10.1083/jcb.202010106).
The discovery of the existence of the dual spindle resulted in a broad media coverage across the world and interest by the general public. The impact of this work was highlighted in several German newspapers and magazines (FAZ, Zeit, Süddeutsche, Spektrum der Wissenschaft and EMMA) as well as by a Science perspective article written by Melina Schuh, a Developmental Cell preview article by Michael A. Lampson and a Dispatch in Current Biology article by Marie-Hélène Verlhac. The relevance of our findings for reproductive medicine is furthermore reflected by its influence on ethical discussions especially in Germany. In this context Reichmann et al. was cited in the Leopoldina (German Academy of Science) recommendation for a new law for reproductive medicine presented to the German government in 2021 (https://www.leopoldina.org/uploads/tx_leopublication/2021_Stellungnahme_Embryonenschutz_web.pdf).
The discovery of the dual spindle and that parental chromosomes are separately segregated during the first zygotic division, and the consequent finding that a dual spindle also is frequently present in the bovine embryo was an highly unexpected finding that has strong implications both biologically, and for applicable law and definitions of the first stages of mammalian life in general, and human life specifically. The subsequent molecular studies represent novel ground work and require continuous establishment of new molecular tools and procedures as the previous studies were technically limited by the microscopes available to conduct high-resolution studies in highly light-sensitive specimens, such as the early moue embryo. The first-generation light-sheet microscope developed in our group was the first device enabling such studies and is now complemented by the second generation, the iSPIM2, as a new, even more sensitive light-sheet microscope which has been validated for multiple samples including organoids and mouse embryos providing unprecedented spatio-temporal resolution in highly sensitive samples. The research results described above are the first to unravel in molecular detail the underpinnings of cell division in the very early stages of embryonic development and provide the basis to explain why those stages are so error-prone, giving rise to working hypotheses driving research over many years to follow.