Team
Team manager: Pituello Fabienne
Presentation
Building an organ or preserving its homeostasis requires a tight control of the transition between proliferation and differentiation of stem/progenitor cells. During nervous system development, the temporality of this switch conditions neuron production. Disrupting its timing can lead to several neuro-pathologies such as microcephaly if the progenitor pool is depleted ahead of time or tumours if progenitor cells remain proliferating. Yet, how time is integrated in controlling neurogenesis remains elusive. The concept we investigate is that the cell cycle times the shift from proliferation to differentiation during neurogenesis.
In the developing spinal cord (chicken and mouse), we previously showed that the CDC25B phosphatase, a regulator of the cell cycle G2/M transition, induces a progressive shift from proliferative to differentiating neurogenic divisions using cell cycle independent and dependent mechanisms (Bonnet et al., elife, 2018). Single-cell imaging of the cell cycle reveals that CDC25B shortens the G2 phase and indirectly increases heterogeneity in duration of the G1 phase, a generic feature associated with progenitor maturation and differentiation (Molina et al., Development, 2022). In mouse neocortex, CDC25B also promotes progenitor maturation via the control of G2 phase duration (Roussat et al, J. Neurosci, 2023). Our goal is now to decipher the molecular mechanisms acting downstream of CDC25B and/or of a change in duration of G1 or G2 phases leading to differentiation.
Project 1
Sophie Bel-Vialar, PI
Collaborations at CBI: Simon Lebaron; Célia Plisson-Chastang, team oncorib, MCD; BigA core facility; METI core facility
We recently shed light on the importance of G2 phase kinetics in controlling the transition between proliferation and differentiation of neural progenitor cells. We showed that shortening the G2 phase, both by genetic manipulations or pharmacological treatments, is sufficient to change the neural progenitors’ outcome of spinal and cortical progenitors promoting neuronal differentiation (Roussat et al., 2023 and unpublished data). These results suggest that important regulatory events take place during the G2 phase of the mother cell to control the cellular fate of daughter cells after cell division. By combining several approaches (RNA-Seq, transmission electronic microscopy, Ribo MegaSEC), we obtained data suggesting a link between G2 phase kinetics, the regulation of ribosome biogenesis and translation machinery during the proliferative to neurogenic transition. Together, these original results open the hypothesis that cell cycle dynamics could control the proliferative/neurogenic status of neural progenitors by modulating ribosome homeostasis, thus triggering the differential expression of key proteins for the transition between these two states. We are now investigating in more details this hypothesis using the chick and mouse developing spinal cord as a model.
Project 2
Eric Agius, PI; Odile Mondésert
Collaboration: Proteomic core facility, IPBS Toulouse
CDC25B expressed by proliferating neural progenitor cells stimulates their differentiation into neurons by promoting asymmetric neurogenic divisions giving rise to a progenitor and a neuron, and terminal neurogenic divisions producing two neurons. While CDC25B interaction with its canonical substrates, CDKs, is needed to stimulate symmetric neurogenic divisions, a point mutated form of CDC25B retaining its phosphatase activity but unable to interact with CDKs still promotes asymmetric neurogenic divisions. This CDK-independent mechanism implies the existence of unknown substrates for CDC25B (Bonnet et al, 2018). One of our aims is to identify such substrates for the phosphatase CDC25B. We use a proximity-based labeling technique allowing collecting CDC25B-interacting proteins and mass spectrometry to identify CDC25B interactome-network. (coll. Proteomic facility, IPBS Toulouse). Biochemical and functional tests will be then set up to validate and identify novel CDC25B substrates involved in neurogenesis.
Project 3
Valérie Lobjois, PI; Odile Mondésert; LITC core facility
We recently developed a novel 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. Using this strategy, we demonstrated heterogeneity in cell cycle duration primarily explained by heterogeneity in the length of the G1 phase. Moreover, overexpression of CDC25B increases the variability and length of the G1 phase, which is associated with tissue maturation and differentiation(Molina et al., 2022).
Our aim is to study the mechanisms that control the variability of the G1 phase duration 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 cell cycle. Recent work 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 cell cycle (Min et al., 2020 doi: 10.1126/science.aay8241). Our hypothesis is therefore that heterogeneity in G1 length 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 neural progenitor cells. In order to test this hypothesis, we adapted a reporter to the chicken neural tube model to measure 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 fate of mitosis-derived daughter cells will allow determining the link between variability in the timing of R-point crossing and the fate of mitosis-derived daughter cells. It will also give a track to decipher the molecular mechanisms by which CDC25B, by regulating the duration of the G2 phase, can induce a delay in the timing of the R-point and thereby times the shift from proliferation to differentiation.
Project 4
Eric Agius, PI; Valérie Lobjois
Collaboration J. Gautrais CRCA/CBI and S. Cussat-Blanc IRIT/UTCapitole
Our data suggest that CDC25B expression in the neural progenitors act as a maturating factor, inducing a progressive shift from proliferative to neurogenic divisions. While the prevalent scenario is spinal progenitors constitute a homogeneous population with stochastic mode of division, our data call this homogeneity into question. Theoretical work performed in collaboration with J. Gautrais sustains an alternative hypothesis where the population of neural progenitor cells is heterogeneous with a progressive loss of proliferative capacity at the cell scale (Azais et al., 2019). Preliminary modeling results indicate that the two models are discriminative when considering the distributions of progenitors/neurons’ content within clones. We are currently performing theoretical clonal estimations to compare them to experimental clonal analyses by using the ISiCell platform (ALIFE 2024 proceedings, https://doi.org/10.1162/isal_a_00769). This work will allow discriminating the two models.
Combined with our experimental data, modeling should here be a way to help understanding the mechanisms controlling the transition between proliferation and differentiation of neural progenitor cells, which most likely will be applicable to other stem cells including human neural stem cells.
– Roussat, M., Jungas, T., Audouard, C., Omerani, S., Medevielle, F., Agius, E., Davy, A., Pituello, F., and Bel-Vialar, S. (2023). Control of G2 Phase Duration by CDC25B Modulates the Switch from Direct to Indirect Neurogenesis in the Neocortex. J Neurosci 43, 1154-1165. 10.1523/JNEUROSCI.0825-22.2022.
– Molina, A., Bonnet, F., Pignolet, J., Lobjois, V., Bel-Vialar, S., Gautrais, J.*, Pituello, F.*, and Agius, E.* (2022). Single-cell imaging of the cell cycle reveals CDC25B-induced heterogeneity of G1 phase length in neural progenitor cells. Development 149. 10.1242/dev.199660. *co-corresponding
– Azais, M., Agius, E., Blanco, S., Molina, A., Pituello, F., Tregan, J.M., Vallet A. and Gautrais, J. (2019) Timing the spinal cord development with neural progenitor cells losing their proliferative capacity: a theoretical analysis. Neural Dev 14,7
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– Bonnet, F., Molina, A., Roussat, M., Azais, M., Vialar, S., Gautrais, J., Pituello, F. * and Agius, E. * (2018). Neurogenic decisions require a cell cycle independent function of the CDC25B phosphatase. Elife. Jul 3;7. pii: e32937. doi: 10.7554/eLife.32937 *co-corresponding
– Lacomme, M., Liaubet, L., Pituello, F.* and Bel-vialar, S*. (2012). NEUROG2 drives cell cycle exit of neuronal precursors by specifically repressing a subset of cyclins acting at the G1 and S phases of the cell cycle. Mol Cell Biol 32, 2596-607. *co-corresponding
Affiliation
