Periodic Reporting for period 1 - DualSpindleAssembly (Defining the mechanism of the dual spindle assembly and alignment in the mammalian zygote)
Reporting period: 2021-06-01 to 2023-05-31
The development of mammalian life begins with fertilization, the fusion of two haploid cells, oocyte and sperm, which results in the formation of the diploid zygote containing two pronuclei. For the faithful development of an organism, maintenance of its genomic integrity during early embryonic mitosis is essential as genetic abnormalities transmitted through the blastomeres can result in pregnancy failures and/or severe fetal disorders. Given its crucial importance, cell division is surprisingly error-prone at the beginning of mammalian life. The reported incidence of aneuploidy (presence of an abnormal number of chromosomes in a cell) in early human embryo exceeds 50% and is a major cause of infertility and severe congenital disorders.
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 thought that a single spindle combines the maternal and paternal chromosomes. However, it is recently shown that two bipolar spindles form in the zygote which independently congress the maternal and paternal chromosomes and then must be aligned to execute the first division faithfully. This intriguing spindle assembly revealed the mechanism behind the long-standing observation that parental genomes occupy separate nuclear compartments in the two cell embryo and provides a likely rationale for erroneous divisions into more than two blastomeric nuclei observed in other mammalian zygotes, including human. Indeed, preventing the alignment of the two spindles after the assembly gives rise to multi-nucleated two-cell embryos in mouse. In mouse zygotes the alignment is rather reliable if not artificially perturbed, but in zygotes of other mammalian species it seems to be much less faithful. While the discovery of the dual spindle in mammalian zygotes has provided important new basic insights into the first mitotic division, the molecular mechanisms underlying dual spindle formation are still elusive.
In this project, I established functional live-imaging analysis for the mouse zygote by combining advanced light sheet technics and molecular perturbations. With this approach, I was able to dissect the molecular mechanisms of dual spindle assembly and alignment in the first division. Indeed, I identify the major microtubules (MTs) nucleation sites and pathways responsible for dual spindle formation. Moreover, I determined the molecule essential for dual spindle alignment. Together with detailed molecular analysis of the molecules I identified, I established a new molecular model for the dual spindle formation in the mouse zygote.
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 thought that a single spindle combines the maternal and paternal chromosomes. However, it is recently shown that two bipolar spindles form in the zygote which independently congress the maternal and paternal chromosomes and then must be aligned to execute the first division faithfully. This intriguing spindle assembly revealed the mechanism behind the long-standing observation that parental genomes occupy separate nuclear compartments in the two cell embryo and provides a likely rationale for erroneous divisions into more than two blastomeric nuclei observed in other mammalian zygotes, including human. Indeed, preventing the alignment of the two spindles after the assembly gives rise to multi-nucleated two-cell embryos in mouse. In mouse zygotes the alignment is rather reliable if not artificially perturbed, but in zygotes of other mammalian species it seems to be much less faithful. While the discovery of the dual spindle in mammalian zygotes has provided important new basic insights into the first mitotic division, the molecular mechanisms underlying dual spindle formation are still elusive.
In this project, I established functional live-imaging analysis for the mouse zygote by combining advanced light sheet technics and molecular perturbations. With this approach, I was able to dissect the molecular mechanisms of dual spindle assembly and alignment in the first division. Indeed, I identify the major microtubules (MTs) nucleation sites and pathways responsible for dual spindle formation. Moreover, I determined the molecule essential for dual spindle alignment. Together with detailed molecular analysis of the molecules I identified, I established a new molecular model for the dual spindle formation in the mouse zygote.
First, I set up culture and micromanipulation conditions for the mouse zygote. Subsequently, I used the inverted light sheet microscopy (SPIM) built in the Ellenberg group and established functional live-imaging analysis for the mouse zygote combining molecular perturbations. This opens up new research opportunities and contributes to establish the early mouse zygote as a powerful model system for molecular cell biology. Second, I determined the major MT nucleation site and pathway for dual spindle assembly using functional live-imaging analysis. Third, I identified the molecule required for dual spindle alignment. Together with detailed molecular analysis of the molecules I identified, I established a new model for the dual spindle assembly and alignment in the mouse zygote. Finally, I will check if errors in spindle formation are the cause of parental genome loss by validating key molecules in model organisms with different alignment fidelities. Thus, my studies will improve our understanding of cell division in mammals and human infertility.
At two international conferences, I communicated my ongoing results and interacted with other international researchers in the field. All the analysis will be completed within the next three months so that the manuscript covering all my results will be ready for submission in six months. After submission, I will also communicate my results and experience to different target audiences via outreach activities.
At two international conferences, I communicated my ongoing results and interacted with other international researchers in the field. All the analysis will be completed within the next three months so that the manuscript covering all my results will be ready for submission in six months. After submission, I will also communicate my results and experience to different target audiences via outreach activities.
A high aneuploidy rate has been reported in early embryos and is a major cause of miscarriages. Therefore, beyond the fundamental knowledge gain, this project is of high importance to generate molecular hypotheses for the causes of human infertility and congenital diseases. My results of the project in the long-term could not only advance the field in human reproductive medicine but also contribute to the agro-industry section for livestock maintenance by the improvement of the success rate of the in vitro fertilization (IVF) process. To achieve my project, I pushed the technical boundaries during this project. By adapting the novel light sheet microscopy to my research I established light sheet microscopy for highest resolution live-embryo imaging which provides several opportunities to unravel molecular processes in living mouse embryos and makes the microscope the perfect tool for understanding the mechanisms of dual spindle assembly in mammalian zygotes. Combining perturbation assays with the high-resolution imaging to disrupt and directly observe multiple protein function in live early embryos opened up new research opportunities and contribute to establish the early mouse embryo as a powerful model system for molecular cell biology. The image data and methods will be made publicly available to provide valuable tools and resources to the scientific community.
Thus, I established innovative cutting-edge technologies in the mammalian embryo during this project, advanced the field of early mammalian development and understanding human infertility and provided knowledge, tools and data resources to the community to promote research excellence.
Thus, I established innovative cutting-edge technologies in the mammalian embryo during this project, advanced the field of early mammalian development and understanding human infertility and provided knowledge, tools and data resources to the community to promote research excellence.