Periodic Reporting for period 4 - CHROMONUMBER (Chromosome number variations in vivo: probing mechanisms of genesis and elimination)
Periodo di rendicontazione: 2022-01-01 al 2023-06-30
Most cells in animals have a euploid stable genome content. This is important to guarantee genetic stability over many generations. Most animal cells are diploid, which were inherited by the parental genomes. Deviations to the euploid genome can be found in healthy organisms. This is the case of 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. Aneuploidy is at the basis of several human diseases. This is the case of many different human cancers with genetic instability.
This project provided the ground to establish novel concepts and avenues or research.I consider that this project was quite impactful on many fronts. For example the unexpected findings in aim 1 are very important for the whole fly community as they revealed the presence of unknown gene regulatory mechanisms in the developing fly brain. The findings of aime 2 have the potential to inform us and the community of the origins of the vulnerability of the mammalian stem cell population and so how these can help us understand he origins of brain growth disorders. Finally, aim 3 set the stage to understand the consequences of unscheduled whole genome duplications, which is important as this is the second most frequent condition in human tumors.
This project provided the ground to establish novel concepts and avenues or research.I consider that this project was quite impactful on many fronts. For example the unexpected findings in aim 1 are very important for the whole fly community as they revealed the presence of unknown gene regulatory mechanisms in the developing fly brain. The findings of aime 2 have the potential to inform us and the community of the origins of the vulnerability of the mammalian stem cell population and so how these can help us understand he origins of brain growth disorders. Finally, aim 3 set the stage to understand the consequences of unscheduled whole genome duplications, which is important as this is the second most frequent condition in human tumors.
1) Related to aim1, we wanted to generate a genetic instability fluorescent sensor based on the expression of green fluorescence protein (GFP) when a given chromosome was lost. We generated 25 Drosophila lines, where the sensor cassette was inserted at different locations of all fly chromosomes. Initially screen revealed very low levels of green cells, explained by the fact that in wild type individuals, the levels of chromosome loss were very low. However, when we analysed the developing brain, we realized that these in this tissue the frequency of green cells was very high. We spent a lot of time characterizing these findings to conclude that the origin of green cells were not explained by chromosome loss. Instead we found a novel type of gene expression regulation that in a stochastic manner lead to the expression of GFP. We called this phenomenon illuminati and we further showed that illuminati is influenced by environmental factors such as temperature or food composition. We initially posted these findings in a form of an article in Biorx and we were contacted by the lab of Dr C. Gonzalez at the IRB, in Barcelona, Spain. His lab had found very similar results suing other type of sensors. We published in 2022 an original article in the review Development, describing the findings of both labs (Goupil et al, 2022).
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 published in Current Biology (Vargas et al, 2019). We have continued to characterize other factors that can influence mitotic spindle assembly and accurate chromosome segregation in the developing mouse brain. We have found that the cellular cortex behaves in different ways at these two developmental stages with key differences in composition and mechanical properties. These results are currently being prepared to be published in a form of an article.
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 cells show high levels of DNA damage. The reasons behind these defects have further been investigated. We have found that newly born tetraploid cells fail to scale content size and to adapt to DNA increase, which leads to significant defects in DNA replication and replication stress. Importantly, single cell sequencing revealed very high levels of genetic instability. These results were published last year - Gemble et al, 2022 Nature. Still related with the progression of polyploid cells along the cell cycle we have found that as they enter mitosis, cells fail to undergo bipolar division. These cells have extra centrosomes and chromosomes and so by using γ live imaging approaches, we noticed that centrosomes fail to coalesce to form a bipolar spindle. This seems to be related with steric hindrance imposed by the increase in DNA content. We used mathematical simulations to built a new model of centrosome behaviour during mitosis. These results have been published in 2020- Goupil et al, JCB.
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 published in Current Biology (Vargas et al, 2019). We have continued to characterize other factors that can influence mitotic spindle assembly and accurate chromosome segregation in the developing mouse brain. We have found that the cellular cortex behaves in different ways at these two developmental stages with key differences in composition and mechanical properties. These results are currently being prepared to be published in a form of an article.
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 cells show high levels of DNA damage. The reasons behind these defects have further been investigated. We have found that newly born tetraploid cells fail to scale content size and to adapt to DNA increase, which leads to significant defects in DNA replication and replication stress. Importantly, single cell sequencing revealed very high levels of genetic instability. These results were published last year - Gemble et al, 2022 Nature. Still related with the progression of polyploid cells along the cell cycle we have found that as they enter mitosis, cells fail to undergo bipolar division. These cells have extra centrosomes and chromosomes and so by using γ live imaging approaches, we noticed that centrosomes fail to coalesce to form a bipolar spindle. This seems to be related with steric hindrance imposed by the increase in DNA content. We used mathematical simulations to built a new model of centrosome behaviour during mitosis. These results have been published in 2020- Goupil et al, JCB.
In a few words our project went well beyond the state-of-the-art. We have generated novel findings that were not predicted. These include the existence of a novel phenomenon- Illuminati in the fly brain that is extremely important for the community. With the analysis of neurogenesis in the mammalian brain we have conceptualised that the same cell type can have different modes of spindle assembly and different capacity to induce chromosome segregation errors dependent on the expression of key microtubule proteins and the actin cortex. Finally, we have found that aneuploidy and polyploidy can have divergent consequences according to the cell of origin. Moreover, our work has shown for the first time that scaling defects are at the origin of genetic instability.