A fundamental issue in developmental biology and in nervous system homeostasis is to understand the mechanisms governing the balance between maintenance of proliferating neural progenitor cells versus their differentiation into post-mitotic neurons. Alterations in that balance could result in neurodevelopmental disorders or stem cell-related brain tumorigenesis following oncogenic transformation. The discovery of cell reprogramming and the prospect of stem cell based therapy highlight the importance of elucidating the mechanisms controlling this fine balance in normal and pathological settings.
Changes in cell cycle of neural progenitor cells (NPC) have been proposed as one of the mechanisms triggering the decision from proliferation to differentiation (Agius et al, 2015; Molina & Pituello, 2016). However, a comprehensive model for this process is still lacking and whether changes in cell cycle kinetics of NPC cause neurogenesis or whether both events are regulated in parallel is still a matter of debate. This caveat is related to the fact that these analyses were mainly performed on fixed tissue and/or in heterogeneous populations of progenitors performing proliferative and differentiating divisions that are spatially intermingled and indistinguishable without lineage tracing. The main goal of our project was to elucidate in living cells whether and how changes in cell cycle kinetics, switch a proliferating neural progenitor into a differentiating neuron.
To help us accomplish this goal we recently set up a novel high resolution time-lapse imaging technique that allows measuring the duration of each phase of the cell cycle in single neural progenitors in the chicken developing neural tube and tracking the fate of daughter cells after mitosis. We engineered a cell cycle sensor allowing an unambiguous detection of the four cell cycle phases. It comprises: -1) a fluorescently tagged proliferating cell nuclear antigen (PCNA-GFP) displaying uniform distribution in G1 and G2 nuclei, dots in S phase nuclei and a diffuse distribution in M phase cells upon nuclear envelope breakdown; -2) the Fucci probe derived from Cdt1 (zpFucci-G1 Orange) which labels G1 nuclei in orange and persists in G0 nuclei in differentiating neurons. This sensor is electroporated in the chicken neural tube to reproducibly obtain a high degree of mosaicism compatible with lineage tracing. To assign cell cycle kinetics to a specific cell fate, we employ long-term, high resolution time-lapse imaging of single cells using confocal microscopy.
Using this strategy, we have demonstrated variability in cell cycle duration and in particular in G1 phase duration in the neural progenitor population. Moreover, overexpression of CDC25B increases the variability of G1 phase duration (Molina et al., 2022).
Our aim is to study the mechanisms that control the variability of the duration of the G1 phase and to determine its role in the spatio-temporal regulation of neuronal differentiation. Data from different models show that a progressive lengthening of the G1 phase of the cell cycle of stem or progenitor cells is associated with their differentiation. Progression through the G1 phase is controlled by the passage of the restriction point (R-point), marking the definitive commitment of cells to the cycle. Recent work (Min et al., 2020) in cultured epithelial cells has shown that the integration of signals during the G2 phase of the cell cycle of the mother cell can lead to a passage of the R-point upon exit from mitosis, determining the fate of daughter cells in the next cycle. Our hypothesis is therefore that heterogeneity in the duration of the G1 phase is associated with heterogeneity in the timing of the restriction point passage determined by the duration of the G2 phase of the mother cell in the neural progenitor population. In order to study this heterogeneity, we adapted a reporter to the chicken neural tube model that allows the measurement of the timing of the restriction point during the G1 phase at the level of individual progenitors in vivo by video microscopy. The combination of this reporter with lineage experiments to identify the identity of mitosis-derived daughter cells will allow us to define the link between variability in the timing of R-point crossing and the fate of mitosis-derived daughter cells. This approach will also allow us to study how CDC25B, by regulating the duration of the G2 phase, can induce a delay in the timing of the R-point transition to G1.