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 he
lp 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. Following this approach, it is then possible to identify the cell cycle features of proliferative divisions versus neurogenic divisions and, combined with gain and loss of function experiments, to determine how modifications in cell cycle impact cell fate decision. Furthermore, tracking analysis includes the description of oscillatory nuclear movement known as Interkinetic Nuclear Migration, occurring in phase with the cell cycle, which could be linked to cell fate choice.
Fabienne PITUELLO: fabienne.pituello@univ-tlse3.fr
Angie MOLINA: angie.molina@univ-tlse3.fr
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