Encoding time in neurogenesis: insights into the cell cycle – ETIN-CC

To carry out this work, we used two classic models for studying embryonic development: chickens and mice. The chicken embryo is easily accessible in the egg and can be used to film cell behaviour in real time. It is also a useful model for conducting experiments to track cells and their descendants, as well as gene loss or overexpression. Large quantities can be obtained for large-scale analyses. Mice offer the possibility of using mutants of the genes of interest. We already have a good understanding of the mechanisms of neurogenesis (the birth of neurons) in both models, including the temporal evolution of the mode of division of neural progenitors, which is essential for our study. We conducted our research on the caudal region of the neural tube in chicken and mouse embryos — the future spinal cord — and during the development of the neocortex in mice.

To achieve our objectives, we implemented several complementary approaches:

At the cellular level, we developed a labelling technique for the four phases of the cell cycle in chicken embryos. This was combined with a microscopy imaging technique that enables the real-time measurement of the duration of each phase of the cell cycle, as well as the subsequent fate of the resulting cells (progenitors or neurons).

At the molecular level, we performed comprehensive analyses to identify all genes whose expression is altered in response to CDC25B activity. These analyses led us to hypothesise that protein synthesis could be altered downstream of CDC25B, prompting us to develop strategies to measure this alteration. Finally, we adapted a recent technique for identifying CDC25B interactors in order to find new substrates for the enzyme.

We studied the consequences of CDC25B loss of function on neocortex development in mice using a mutant that we generated.

In collaboration with our partner, we developed a mathematical model to determine whether neural progenitors constitute a homogeneous population with random mitotic index (MoI), or a heterogeneous population in which progenitors derived from progenitor neuron (PN) division lose their proliferative capacity and produce only PN and neural network (NN) divisions, but no more progenitor neuron (PN) divisions. We performed theoretical clonal estimates and experimental clonal analyses to discriminate between these two models.

This study has enabled us to obtain original results regarding the control of the transition from a proliferating neural progenitor cell to a differentiating neuron that has ceased to proliferate — a pivotal stage in the development of a functional nervous system. In the neural tube (the embryonic precursor of the spinal cord), we demonstrate that neural progenitor cells that continue to proliferate enter a new cell cycle as soon as they exit mitosis. We hypothesise that the decision to continue proliferating is made before division, potentially during the G2 phase of the mother cell. Using our real-time imaging strategy, we reveal that the phosphatase CDC25B, active in the G2 phase, indirectly induces elongation of the G1 phase in neural progenitors. Lengthening of the G1 phase is a common feature of the maturation and differentiation of stem and progenitor cells. The expression of CDC25B in neural progenitors correlates with neurogenesis. It induces a shortening of the G2 phase and, indirectly, a lengthening of the G1 phase. This promotes the transition from a proliferating progenitor cell to a differentiating neuron. These data reveal a new mechanism for prolonging the G1 phase during organ development associated with cell differentiation. To further our understanding of this mechanism, we identified the genes modified downstream of CDC25B and demonstrated that protein synthesis is rapidly stimulated in response to phosphatase activity. Thus, the cell cycle could control the timing of neurogenesis by modulating the synthesis of proteins essential for neuronal differentiation. In collaboration with our partner, we have also developed a mathematical model of neural tube development, focusing particularly on the genesis of spinal motor neurons. This model enables us to propose new hypotheses regarding the production of this neuronal population.

Finally, studying neocortex development in a CDC25B mutant mouse revealed that phosphatase is involved in cortical neurogenesis, specifically the maturation of cortical neural progenitors.

These results are an important addition to our understanding of the mechanisms that regulate the balance between the proliferation and differentiation of neural progenitors, and may also be applicable to the development of other organs.

Motor neurons differentiate in the spinal cord. This population is affected by many diseases, such as Charcot's disease. A key challenge in current research is generating motor neurons from human stem cells derived from patients. The aim is to obtain sufficient quantities of material to better understand the mechanisms involved in neuronal death and to test therapeutic strategies that promote survival. In this context, knowledge of the mechanisms controlling the proliferation/differentiation balance of stem/progenitor cells is clearly of interest, as is the subject of this study.

Deregulation of CDC25B has been associated with many cancers. Data obtained from our embryonic models prompted us to initiate a programme to study the impact of CDC25B on the duration of the G1 phase in glioblastomas.

Finally, we were contacted by a team of geneticists who had identified a mutation in the CDC25B gene in humans. This mutation results in a homozygous state with a distinct microcephaly phenotype accompanied by intellectual disability. We have created a mouse line that exhibits this mutation. Preliminary results suggest that these mice could exhibit a microcephaly phenotype, providing a new model for studying a pathology associated with a neurodevelopmental defect.

Timing the transition between proliferating neural progenitor cells (NPCs) and differentiating neurons is critical for nervous system development and homeostasis. Premature differentiation leads to precocious depletion of the progenitor’s pool associated with neurodevelopmental disorders. It is increasingly apparent that the decision for a cell to exit the cell cycle and differentiate is a consequence of events that have occurred during previous cell cycles. The hypothesis we will investigate is that the cell cycle machinery controls the timing of neurogenesis. Using the developing spinal cord as a paradigm (chicken and mouse), partner 1 recently showed that the CDC25B phosphatase, a regulator of the cell cycle G2/M transition, acts as a maturating factor, inducing asymmetric neurogenic divisions (PN) and neurogenic terminal symmetric divisions (NN) by two distinct molecular mechanisms that remains to be elucidated. We will combine a novel live imaging technique we recently set up, global approaches (transcriptomics, proteomics), functional analyses and mathematical modeling to elucidate how the cell cycle machinery, and particularly the CDC25B phosphatase acts as molecular clock to switch a proliferating neural progenitor into a differentiating neuron.

The project is subdivided in three specific objectives:

1) Deciphering the role of the cell cycle in CDC25B driven terminal neurogenic divisions.
CDC25B promotes NN divisions in a CDK dependent fashion associated with a G1 phase lengthening. We will: i) determine if the G1 phase lengthening is causal to the function of CDC25B on NN divisions (pharmacological and genetic tools); ii) Compare molecular networks modified downstream of CDC25B and following a modification of G1 length (RNAseq); Based on the molecular networks modified downstream of CDC25B and/or G1 lengthening identify and test functionally molecular players promoting terminal neurogenic divisions.

2) Dissecting CDC25B function in promoting asymmetric neurogenic divisions.
CDC25B promotes PN divisions independently of CDK interaction (CDC25BCDK-), which implies an unknown substrate. It also induces fast nuclear movement toward the basal side in early G1 independently of CDK. We plan to i) identify new substrates of CDC25B by a proteomic approach and validate them functionally; ii) Define the sub-cellular localization of CDC25B involved in its neurogenic function (HA-tagged-CDC25B CRISPR/Cas9; CDC25B-GFP); iii) Determine if the change in nucleus movement is causal for the change in the mode of division (MoD) (pharmacologic tools and dominant negative form of molecular motors); use RNAseq data to identify the molecular and signaling pathways modified downstream of CDC25BCDK-.

3) Determining whether the population of progenitors is homogeneous with stochastic MoD or heterogeneous with restricted MoD.
Our data suggest that CDC25B expression in the NPCs acts as a maturating factor, inducing a progressive shift from proliferative (PP) to neurogenic (PN, NN) divisions. if the prevalent scenario is that NPCs constitute a homogeneous population with stochastic MoD, theoretical work performed by partner 2 sustains an alternative hypothesis where NPCs issued from a PN division lose their proliferative capacity, and thereafter produce only PN and NN divisions, but no more PP divisions. Preliminary modeling results indicate that the two models can be discriminated when considering the distribution of progenitors/neurons’ content within clones. We will perform theoretical clonal estimations and compare them to experimental clonal analyses to discriminate between models. Data obtained from the two first objectives will also fuel the model.
We expect to uncover a novel mechanism controlling the transition between NPCs maintenance and differentiation, which most likely will be applicable to other stem cells including human neural stem cells.