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SAC robustness in the transition from meiosis to mitosis

Periodic Reporting for period 1 - SAC_EarlyEmbryo (SAC robustness in the transition from meiosis to mitosis)

Reporting period: 2016-02-01 to 2018-01-31

Maintenance of genomic integrity during meiosis and early embryogenesis is essential as chromosomal and genetic abnormalities transmitted through the gametes or blastomeres can result in pregnancy failures and/or severe fetal disorders. Although in somatic mitotic cells chromosome segregation is extremely trustworthy, different studies in mammals have shown that chromosome segregation errors are strikingly high in early embryonic development, estimating that between 22-80% of the pre-implantation embryos have cells with chromosomal abnormalities. This data suggests that the regulatory mechanisms that ensure faithful chromosome segregation that are very accurate in somatic cells, most importantly the spindle assembly checkpoint (SAC), may behave differently in the first divisions of the embryo.

The SAC is a surveillance mechanism that monitors the presence of unattached chromosomes and inhibits anaphase onset until all the chromosomes are properly attached to microtubules. The relevance and dynamics of the SAC, safeguarding genome integrity, in dividing somatic cells have been deeply studied and it is well established that defects in this checkpoint leads to chromosome missegregation.

However, despite the importance for early embryonic development, the sensitivity of mammalian embryos to light has precluded real-time imaging of chromosome segregation and its control in the first embryonic divisions. Recent advances in light sheet microscopy in the Ellenberg lab have now overcome this limitation. Taking advantage of this unique opportunity I have studied how chromosome segregation takes place during the first embryonic divisions.
First, in order to analyze the SAC function and robustness, first of all I decided to evaluate whether the checkpoint was functional at all during early development. To test it, I activated the checkpoint using the microtubule-depolymerizing agent nocodazole and, therefore, preventing the attachment of microtubules to the kinetochores. This drug induced a long prometaphase arrest in the embryos, as expected in a SAC proficient context. However, the first three cleavages differ in their SAC response, with a longer arrest in the zygote, suggesting an increased sensitivity to the nocodazole treatment. In all cases, the arrest was rescued by the SAC inhibitor reversine, confirming that the arrest is SAC-dependent. In addition, I could detect the recruitment of SAC proteins to the kinetochores in the presence of nocodazole.

Next, I evaluated the impact of inhibiting the SAC on the duration of mitosis. In order to do that I did a dose-response analysis, using several concentration of the SAC inhibitor reversine in the first three divisions of the embryo. The results showed that each stage displays different sensitivity to the inhibition of the SAC. The zygote is less sensitive to SAC inhibition than latter stages, since higher concentrations of reversine are required to have a similar effect than in the 2-Cell or 4-Cell embryos.

These two results combined suggests that the checkpoint, even though functional during the pre-implantation devlopment, is quite robust in the zygote but it becomes weaker in the following divisions.

In addition, with the help of the Gene Editing & Embryology Facility (EMBL-Monterotondo) I just generated the first SAC mouse reporter (Mad2-mEGFP KI) that allows to evaluate the state of the checkpoint by live imaging. Since the process of generating a transgenic mice takes a long time, in parallel, I decided to study the SAC satisfaction using an indirect readout. This checkpoint inhibits the activation of the Anaphase-promoting complex (APC/C), an E3 ubiquitin ligase that targets proteins for degradation.

Therefore, I could identify the inactivation of the SAC indirectly by measuring the timing at which the degradation of its substrates starts. I used a fluorescently tagged version of the APC/C substrate Securin (Securin-EGFP) to measure APC/C activity by quantifying the fluorescent signal in the cytoplasm during the division. This strategy permitted me not only to define the SAC satisfaction timing but also quantify the APC/C activity dynamics, showing that Zygote, 2-Cell and 4-Cell embryo exhibit differences in their APC/C activity. The degradation of APC/C substrates in the zygote takes a long time (over 40-45 minutes) and it gets faster in the next divisions.

To characterize further the APC/C activity I used the APC/C small molecule inhibitor ProTAME. Interestingly, I found out that the long metaphase arrest induced by the drug led to lose of cohesion in the 2-cell and 4-cell embryos but not in the zygote, suggesting that there is something fundamentally different between these stages regarding sister chromatid cohesion (figure 1).

Considering that during the first embryonic divisions takes place the switch from translational to transcriptional control of protein expression, finally I also studied by Western Blot the protein levels of Mad2 and the major APC/C substrates in the metaphase-to-anaphase transition (Cyclin B and Securin) during the first three divisions of the embryo. The levels of these three important proteins progressively decreased (being the Separase inhibitor Securin the most dramatic one), which could explain, at least partially, the different phenotypes previously mentioned: weakening of the SAC, associated to the reduced levels of SAC proteins such as Mad2; strengthening of the effective APC activity, as a result of the decreased levels of the APC/C substrates; and loss of cohesion upon long metaphase arrest, probably influenced by the big drop in Securin levels.
This project has greatly improved our understanding of chromosomes segregation during mammalian pre-implantation development, and it has uncovered potential mechanisms involved in the accumulation of errors occurring ¬in the first embryonic divisions. Therefore, in the long-term the results of my research could eventually have an impact in human health and in vitro fertilization.

Additionally, as previously mentioned, I have generated the first SAC reporter. This mouse model, in the near future, will be an invaluable tool in different fields, such as cell division and cancer research, and therefore will contribute to expand further our understanding of how the SAC behaves in the intact living cell in different contexts.
Example Loss of Cohesion_2CellBastomere