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Chromosome number variations in vivo: probing mechanisms of genesis and elimination

Periodic Reporting for period 2 - CHROMONUMBER (Chromosome number variations in vivo: probing mechanisms of genesis and elimination)

Reporting period: 2019-01-01 to 2020-06-30

Most cells in animals have a euploid stable genome content. This is important to guarantee genetic stability over many generations and to pass on, through the germ line, an adequate genome that can ensure fertilization and viability. Most animal cells are diploid, containing two copies of each chromosome, which were inherited by the parental genomes. Deviations to the euploid genome can be found in healthy organisms. This is the case of polyploidy, where the entire chromosome set is maintained within a cell, or aneuploidy defined by the gain or loss of a single chromosome. How these cells are generated and maintained in homeostasis within the organism is not known. In a way that seems difficult to understand, polyploidy and aneuploidy are also at the basis of several human diseases. This is the case of many different human cancers that show whole genome duplications, which lead to polyploidy and genetic instability, culminating with the generation and maintenance of aneuploid karyotypes. Further, several neuro-developmental disorders are also characterised by chromosome number alterations. In this case, it is thought that the mechanisms responsible for the elimination of abnormal karyotypes efficiently remove these cells from the progenitor population leading to its depletion and consequently to organ size alteration.
The main question addressed in this project relates with understanding the in vivo consequences of chromosome number alterations taking into consideration the mechanisms responsible for generating these alterations and the cellular responses used to eliminate them.
Developing these questions is extremely important as they will lay the basis for a mechanistic understanding of loss of cell homeostasis in response to chromosome number alterations and how these can lead to disease.
The overall objectives of this proposal are:
1) To identify the frequency of aneuploid cells at the level of a whole organism and the underlying mechanisms responsible for their generation;
2) To understand why the brain is such a vulnerable organ in terms of chromosome segregation defects. We model brain growth disorders, known as microcephalies, to ascertain the modes of neural stem cell division;
3) To characterise tissue specific responses to chromosome number alterations. Why and in which way, in certain organs and tissues, cells with abnormal chromosome numbers are maintained within the population while in others they are eliminated or inhibited to proliferate.
1) Related to aim1, we have addressed this question using the fruit fly Drosophila melanogaster. We have generated all the tools required to develop this aim. However, after the initial characterization, we have encountered one unexpected result (please see technical issue section). This has required some troubleshooting and use of other parameters in order to carry this aim forward. This has resulted in a delay related with this aim, but things are now flowing normally.
In terms of the mechanism at stake involved in generation of aneuploid cells from polyploid cells, we have identified that chromosomes function as a barrier limiting spindle pole coalescence. This favours multipolar divisions prone to chromosome segregation errors and consequent aneuploidy. These results are extremely novel and can provide original views to explain abnormal cell division of organs that contain polyploid cells such as the liver or the pancreas. These results have been recently accepted for publication in the form of an article in the Journal of Cell Biology (Goupil et al., in press).
2) Related with aim 2, we have characterised the morphology of the mitotic spindle during neurogenesis using the developing mouse brain as a model system. Neurogenesis spans from embryonic developmental day 10.5 (E10.5) to E18.5 and to initiate this characterization in an unbiased manner, we used super-resolution confocal microscopy to obtain parameters that define the mitotic spindle in apical neuronal progenitors during this period. We identify changes in spindle morphology between early and late developmental stages. At early stages, the mitotic spindle contains long and numerous astral microtubules, the microtubules responsible for establishing interactions with the cortex. Additionally, the mid-spindle region appears less dense, suggesting that the region where chromosomes are normally aligned at the metaphase plate is not as crowded as in other cell types. Importantly, we have also found that late neurogenesis mitotic spindles contain fewer astral microtubules, whereas mid-spindle microtubule density was increased. Thus our work uncovers a novel concept: in the neural stem cell population, the mitotic spindle can undergo morphological changes during embryonic development. To test if different spindle morphologies influence the capacity to segregate chromosomes, we challenged mitosis using two different inhibitors. We found indeed that early neurogenesis spindles generated more mitotic errors. Further we identified TPX2, as an essential molecular player in altering spindle morphology within this context. Our results thus provide a framework to understand the genesis of neuro-developmental disorders like primary recessive microcephaly and the vulnerability of neuronal progenitors to mutations that affect or compromise the function of the mitotic spindle. These results have been recently published in Current Biology (Vargas et al, 2019).
3) Related with aim 3, we have characterized the tissue responses at stake when cells with abnormal chromosome numbers are generated. We have found unexpected differences specific of tissue identity. While non-epithelial neural stem cells from the brain continue to cycle in response to polyploidy and generate DNA damage during mitosis, they undergo premature differentiation in response to aneuploidy (Results recently published in Current Biology- Nano et al, 2019). Further, we have also found that during interphase, polyploid cellsshow high levels of DNA damage. The reasons behind these defects are currently being investigated in the lab. In sharp contrast, in epithelial tissues, both cell types are eliminated by apoptosis. To gain insights on the mechanisms responsible for triggering apoptosis in epithelia, we have analysed, using various imaging techniques these conditions. We have found two unexpected behaviours of polyploid cells when their cell death is inhibited. The first response is a cell-autonomous response, whereby polyploid cells of epithelial origin will lose epithelial identity and start migrating along the tissue of origin and even invading other tissues. The second response, is a non-cell autonomous response. In this case, as the polyploid cells grow and continue to proliferate (after cell death inhibition), they induce high levels of DNA damage in neighbouring cells, while they show little DNA damage. Both responses are completely unexpected and have never been reported before.
We have progressed enormously since the beginning of the project. We wanted to generate chromosome loss probes that can easily be manipulated to detect these type of events in any Drosophila tissue and this has been the case. I predict that this analysis will result in the accumulation of important data that will contribute to our understanding of aneuploidy generation in vivo. Further, we have identified the extra DNA in polyploid cells as a major obstacle to centrosome clustering in polyploid cells, which might have quite important repercussion in cancer research (article in press). We hope that this data can be translated in a high throughput analysis of centrosome and chromosome number in cancer. Many results are still in the process of being obtained (see project achievements in the following section) and these will be included in future publications.

Considering aim2, we have revealed an unexpected switch in spindle morphology during mouse brain development that should open the view for mitotic spindle analysis in different tissues at different stages of development (paper published in 2019). We are currently continuing to develop this aim tackling this question from another point of view, which is related with the contribution of the tissue to spindle morphogenesis and chromosome segregation (paper in preparation).

Related with aim3, we have found that polyploid cells generate DNA damage in mitosis and interphase. This was not known and it is a major focus of the lab. One paper has been recently published and I can predict that related to this aim, by the end of the project at least two other papers will be published.