Control of G2 Phase Duration by CDC25B Modulates the Switch from Direct to Indirect Neurogenesis in the Neocortex.

During development, cortical neurons are produced in a temporally regulated sequence from apical progenitors, directly or indirectly, through the production of intermediate basal progenitors. The balance between these major progenitor types is critical for the production of the proper number and types of neurons, and it is thus important to decipher the cellular and molecular cues controlling this equilibrium. Here we address the role of a cell cycle regulator, the CDC25B phosphatase, in this process. We show that, in the developing mouse neocortex of both sex, deleting CDC25B in apical progenitors leads to a transient increase in the production of TBR1+ neurons at the expense of TBR2+ basal progenitors. This phenotype is associated with lengthening of the G2 phase of the cell cycle, the total cell cycle length being unaffected. Using in utero electroporation and cortical slice cultures, we demonstrate that the defect in TBR2+ basal progenitor production requires interaction with CDK1 and is because of the G2 phase lengthening in CDC25B mutants. Together, this study identifies a new role for CDC25B and G2 phase length in direct versus indirect neurogenesis at early stages of cortical development.SIGNIFICANCE STATEMENT This study is the first analysis of the function of CDC25B, a G2/M regulator, in the developing neocortex. We show that removing CDC25B function leads to a transient increase in neuronal differentiation at early stages, occurring simultaneously with a decrease in basal intermediate progenitors (bIPs). Conversely, a CDC25B gain of function promotes production of bIPs, and this is directly related to CDC25B's ability to regulate CDK1 activity. This imbalance of neuron/progenitor production is linked to a G2 phase lengthening in apical progenitors; and using pharmacological treatments on cortical slice cultures, we show that shortening the G2 phase is sufficient to enhance bIP production. Our results reveal the importance of G2 phase length regulation for neural progenitor fate determination.

Single-cell imaging of the cell cycle reveals CDC25B-induced heterogeneity of G1 phase length in neural progenitor cells.

Although lengthening of the cell cycle and G1 phase is a generic feature of tissue maturation during development, the underlying mechanism remains poorly understood. Here, we develop a time-lapse imaging strategy to measure the four cell cycle phases in single chick neural progenitor cells in their endogenous environment. We show that neural progenitors are widely heterogeneous with respect to cell cycle length. This variability in duration is distributed over all phases of the cell cycle, with the G1 phase contributing the most. Within one cell cycle, each phase duration appears stochastic and independent except for a correlation between S and M phase duration. Lineage analysis indicates that the majority of daughter cells may have a longer G1 phase than mother cells, suggesting that, at each cell cycle, a mechanism lengthens the G1 phase. We identify that the CDC25B phosphatase known to regulate the G2/M transition indirectly increases the duration of the G1 phase, partly through delaying passage through the restriction point. We propose that CDC25B increases the heterogeneity of G1 phase length, revealing a previously undescribed mechanism of G1 lengthening that is associated with tissue development.

Neurogenic decisions require a cell cycle independent function of the CDC25B phosphatase.

A fundamental issue in developmental biology and in organ homeostasis is understanding the molecular mechanisms governing the balance between stem cell maintenance and differentiation into a specific lineage. Accumulating data suggest that cell cycle dynamics play a major role in the regulation of this balance. Here we show that the G2/M cell cycle regulator CDC25B phosphatase is required in mammals to finely tune neuronal production in the neural tube. We show that in chick neural progenitors, CDC25B activity favors fast nuclei departure from the apical surface in early G1, stimulates neurogenic divisions and promotes neuronal differentiation. We design a mathematical model showing that within a limited period of time, cell cycle length modifications cannot account for changes in the ratio of the mode of division. Using a CDC25B point mutation that cannot interact with CDK, we show that part of CDC25B activity is independent of its action on the cell cycle.

FGF signaling controls Shh-dependent oligodendroglial fate specification in the ventral spinal cord.

BACKGROUND: Most oligodendrocytes of the spinal cord originate from ventral progenitor cells of the pMN domain, characterized by expression of the transcription factor Olig2. A minority of oligodendrocytes is also recognized to emerge from dorsal progenitors during fetal development. The prevailing view is that generation of ventral oligodendrocytes depends on Sonic hedgehog (Shh) while dorsal oligodendrocytes develop under the influence of Fibroblast Growth Factors (FGFs). RESULTS: Using the well-established model of the chicken embryo, we show that ventral spinal progenitor cells activate FGF signaling at the onset of oligodendrocyte precursor cell (OPC) generation. Inhibition of FGF receptors at that time appears sufficient to prevent generation of ventral OPCs, highlighting that, in addition to Shh, FGF signaling is required also for generation of ventral OPCs. We further reveal an unsuspected interplay between Shh and FGF signaling by showing that FGFs serve dual essential functions in ventral OPC specification. FGFs are responsible for timely induction of a secondary Shh signaling center, the lateral floor plate, a crucial step to create the burst of Shh required for OPC specification. At the same time, FGFs prevent down-regulation of Olig2 in pMN progenitor cells as these cells receive higher threshold of the Shh signal. Finally, we bring arguments favoring a key role of newly differentiated neurons acting as providers of the FGF signal required to trigger OPC generation in the ventral spinal cord. CONCLUSION: Altogether our data reveal that the FGF signaling pathway is activated and required for OPC commitment in the ventral spinal cord. More generally, our data may prove important in defining strategies to produce large populations of determined oligodendrocyte precursor cells from undetermined neural progenitors, including stem cells. In the long run, these new data could be useful in attempts to stimulate the oligodendrocyte fate in residing neural stem cells.

A long range distal enhancer controls temporal fine-tuning of PAX6 expression in neuronal precursors.

Proper embryonic development relies on a tight control of spatial and temporal gene expression profiles in a highly regulated manner. One good example is the ON/OFF switching of the transcription factor PAX6 that governs important steps of neurogenesis. In the neural tube PAX6 expression is initiated in neural progenitors through the positive action of retinoic acid signaling and downregulated in neuronal precursors by the bHLH transcription factor NEUROG2. How these two regulatory inputs are integrated at the molecular level to properly fine tune temporal PAX6 expression is not known. In this study we identified and characterized a 940-bp long distal cis-regulatory module (CRM), located far away from the PAX6 transcription unit and which conveys positive input from RA signaling pathway and indirect repressive signal(s) from NEUROG2. These opposing regulatory signals are integrated through HOMZ, a 94 bp core region within E940 which is evolutionarily conserved in distant organisms such as the zebrafish. We show that within HOMZ, NEUROG2 and RA exert their opposite temporal activities through a short 60 bp region containing a functional RA-responsive element (RARE). We propose a model in which retinoic acid receptors (RARs) and NEUROG2 repressive target(s) compete on the same DNA motif to fine tune temporal PAX6 expression during the course of spinal neurogenesis.

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.

Mitochondria, long known as the cell powerhouses, also regulate redox signaling and arbitrate cell survival. The organelles are now appreciated to exert additional critical roles in cell state transition from a pluripotent to a differentiated state through balancing glycolytic and respiratory metabolism. These metabolic adaptations were recently shown to be concomitant with mitochondrial morphology changes and are thus possibly regulated by contingencies of mitochondrial dynamics. In this context, we examined, for the first time, mitochondrial network plasticity during the transition from proliferating neural progenitors to post-mitotic differentiating neurons. We found that mitochondria underwent morphological reshaping in the developing neural tube of chick and mouse embryos. In the proliferating population, mitochondria in the mitotic cells lying at the apical side were very small and round, while they appeared thick and short in interphase cells. In differentiating neurons, mitochondria were reorganized into a thin, dense network. This reshaping of the mitochondrial network was not specific of a subtype of progenitors or neurons, suggesting that this is a general event accompanying neurogenesis in the spinal cord. Our data shed new light on the various changes occurring in the mitochondrial network during neurogenesis and suggest that mitochondrial dynamics could play a role in the neurogenic process.

Cell cycle and cell fate in the developing nervous system: the role of CDC25B phosphatase.

Deciphering the core machinery of the cell cycle and cell division has been primarily the focus of cell biologists, while developmental biologists have identified the signaling pathways and transcriptional programs controlling cell fate choices. As a result, until recently, the interplay between these two fundamental aspects of biology have remained largely unexplored. Increasing data show that the cell cycle and regulators of the core cell cycle machinery are important players in cell fate decisions during neurogenesis. Here, we summarize recent data describing how cell cycle dynamics affect the switch between proliferation and differentiation, with an emphasis on the roles played by the cell cycle regulators, the CDC25 phosphatases.

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.

Proneural NEUROG2 (neurogenin 2 [Ngn2]) is essential for neuronal commitment, cell cycle withdrawal, and neuronal differentiation. Although NEUROG2's influence on neuronal commitment and differentiation is beginning to be clarified, its role in cell cycle withdrawal remains unknown. We therefore set out to investigate the molecular mechanisms by which NEUROG2 induces cell cycle arrest during spinal neurogenesis. We developed a large-scale chicken embryo strategy, designed to find gene networks modified at the onset of NEUROG2 expression, and thereby we identified those involved in controlling the cell cycle. NEUROG2 activation leads to a rapid decrease of a subset of cell cycle regulators acting at G(1) and S phases, including CCND1, CCNE1/2, and CCNA2 but not CCND2. The use of NEUROG2VP16 and NEUROG2EnR, acting as the constitutive activator and repressor, respectively, indicates that NEUROG2 indirectly represses CCND1 and CCNE2 but opens the possibility that CCNE2 is also repressed by a direct mechanism. We demonstrated by phenotypic analysis that this rapid repression of cyclins prevents S phase entry of neuronal precursors, thus favoring cell cycle exit. We also showed that cell cycle exit can be uncoupled from neuronal differentiation and that during normal development NEUROG2 is in charge of tightly coordinating these two processes.

Functional effects of PTPN11 (SHP2) mutations causing LEOPARD syndrome on epidermal growth factor-induced phosphoinositide 3-kinase/AKT/glycogen synthase kinase 3beta signaling.

LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3beta (GSK-3beta) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3beta hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3beta stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.

Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord.

BACKGROUND: During the development of the nervous system, neural progenitor cells can either stay in the pool of proliferating undifferentiated cells or exit the cell cycle and differentiate. Two main factors will determine the fate of a neural progenitor cell: its position within the neuroepithelium and the time at which the cell initiates differentiation. In this paper we investigated the importance of the timing of cell cycle exit on cell-fate decision by forcing neural progenitors to cycle and studying the consequences on specification and differentiation programs. RESULTS: As a model, we chose the spinal progenitors of motor neurons (pMNs), which switch cell-fate from motor neurons to oligodendrocytes with time. To keep pMNs in the cell cycle, we forced the expression of G1-phase regulators, the D-type cyclins. We observed that keeping neural progenitor cells cycling is not sufficient to retain them in the progenitor domain (ventricular zone); transgenic cells instead migrate to the differentiating field (mantle zone) regardless of cell cycle exit. Cycling cells located in the mantle zone do not retain markers of neural progenitor cells such as Sox2 or Olig2 but upregulate transcription factors involved in motor neuron specification, including MNR2 and Islet1/2. These cycling cells also progress through neuronal differentiation to axonal extension. We also observed mitotic cells displaying all the features of differentiating motor neurons, including axonal projection via the ventral root. However, the rapid decrease observed in the proliferation rate of the transgenic motor neuron population suggests that they undergo only a limited number of divisions. Finally, quantification of the incidence of the phenotype in young and more mature neuroepithelium has allowed us to propose that once the transcriptional program assigning neural progenitor cells to a subtype of neurons is set up, transgenic cells progress in their program of differentiation regardless of cell cycle exit. CONCLUSION: Our findings indicate that maintaining neural progenitor cells in proliferation is insufficient to prevent differentiation or alter cell-fate choice. Furthermore, our results indicate that the programs of neuronal specification and differentiation are controlled independently of cell cycle exit.

The on/off of Pax6 controls the tempo of neuronal differentiation in the developing spinal cord.

During neurogenesis, complex networks of genes act sequentially to control neuronal differentiation. In the neural tube, the expression of Pax6, a paired-box-containing gene, just precedes the appearance of the first post-mitotic neurons. So far, its only reported function in the spinal cord is in specifying subsets of neurons. Here we address its possible function in controlling the balance between proliferation and commitment of neural progenitors. We report that increasing Pax6 level is sufficient to push neural progenitors toward cell cycle exit and neuronal commitment via Neurogenin 2 (Ngn2) upregulation. However, neuronal precursors maintaining Pax6(On) fail to perform neuronal differentiation. Conversely, turning off Pax6 function in these precursors is sufficient to provoke premature differentiation and the number of differentiated neurons depends of the amount of Pax6 protein. Moreover, we found that Pax6 expression involves negative feedback regulation by Ngn2 and this repression is critical for the proneural activity of Ngn2. We present a model in which the level of Pax6 activity first conditions the moment when a given progenitor will leave the cell cycle and second, the moment when a selected neuronal precursor will irreversibly differentiate.

Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups.

Initiation of Hox genes requires interactions between numerous factors and signaling pathways in order to establish their precise domain boundaries in the developing nervous system. There are distinct differences in the expression and regulation of members of Hox genes within a complex suggesting that multiple competing mechanisms are used to initiate their expression domains in early embryogenesis. In this study, by analyzing the response of HoxB genes to both RA and FGF signaling in neural tissue during early chick embryogenesis (HH stages 7-15), we have defined two distinct groups of Hox genes based on their reciprocal sensitivity to RA or FGF during this developmental period. We found that the expression domain of 5' members from the HoxB complex (Hoxb6-Hoxb9) can be expanded anteriorly in the chick neural tube up to the level of the otic vesicle following FGF treatment and that these same genes are refractory to RA treatment at these stages. Furthermore, we showed that the chick caudal-related genes, cdxA and cdxB, are also responsive to FGF signaling in neural tissue and that their anterior expansion is also limited to the level of the otic vesicle. Using a dominant negative form of a Xenopus Cdx gene (XcadEnR) we found that the effect of FGF treatment on 5' HoxB genes is mediated in part through the activation and function of CDX activity. Conversely, the 3' HoxB genes (Hoxb1 and Hoxb3-Hoxb5) are sensitive to RA but not FGF treatments at these stages. We demonstrated by in ovo electroporation of a dominant negative retinoid receptor construct (dnRAR) that retinoid signaling is required to initiate expression. Elevating CDX activity by ectopic expression of an activated form of a Xenopus Cdx gene (XcadVP16) in the hindbrain ectopically activates and anteriorly expands Hoxb4 expression. In a similar manner, when ectopic expression of XcadVP16 is combined with FGF treatment, we found that Hoxb9 expression expands anteriorly into the hindbrain region. Our findings suggest a model whereby, over the window of early development we examined, all HoxB genes are actually competent to interpret an FGF signal via a CDX-dependent pathway. However, mechanisms that axially restrict the Cdx domains of expression, serve to prevent 3' genes from responding to FGF signaling in the hindbrain. FGF may have a dual role in both modulating the accessibility of the HoxB complex along the axis and in activating the expression of Cdx genes. The position of the shift in RA or FGF responsiveness of Hox genes may be time dependent. Hence, the specific Hox genes in each of these complementary groups may vary in later stages of development or other tissues. These results highlight the key role of Cdx genes in integrating the input of multiple signaling pathways, such as FGFs and RA, in controlling initiation of Hox expression during development and the importance of understanding regulatory events/mechanisms that modulate Cdx expression.