Neonatal brain injury unravels transcriptional and signaling changes underlying the reactivation of cortical progenitors.

Germinal activity persists throughout life within the ventricular-subventricular zone (V-SVZ) of the postnatal forebrain due to the presence of neural stem cells (NSCs). Accumulating evidence points to a recruitment for these cells following early brain injuries and suggests their amenability to manipulations. We used chronic hypoxia as a rodent model of early brain injury to investigate the reactivation of cortical progenitors at postnatal times. Our results reveal an increased proliferation and production of glutamatergic progenitors within the dorsal V-SVZ. Fate mapping of V-SVZ NSCs demonstrates their contribution to de novo cortical neurogenesis. Transcriptional analysis of glutamatergic progenitors shows parallel changes in methyltransferase 14 (Mettl14) and Wnt/ß-catenin signaling. In agreement, manipulations through genetic and pharmacological activation of Mettl14 and the Wnt/ß-catenin pathway, respectively, induce neurogenesis and promote newly-formed cell maturation. Finally, labeling of young adult NSCs demonstrates that pharmacological NSC activation has no adverse effects on the reservoir of V-SVZ NSCs and on their germinal activity.

The temporal balance between self-renewal and differentiation of human neural stem cells requires the amyloid precursor protein.

Neurogenesis in the developing human cerebral cortex occurs at a particularly slow rate owing in part to cortical neural progenitors preserving their progenitor state for a relatively long time, while generating neurons. How this balance between the progenitor and neurogenic state is regulated, and whether it contributes to species-specific brain temporal patterning, is poorly understood. Here, we show that the characteristic potential of human neural progenitor cells (NPCs) to remain in a progenitor state as they generate neurons for a prolonged amount of time requires the amyloid precursor protein (APP). In contrast, APP is dispensable in mouse NPCs, which undergo neurogenesis at a much faster rate. Mechanistically, APP cell-autonomously contributes to protracted neurogenesis through suppression of the proneurogenic activator protein-1 transcription factor and facilitation of canonical WNT signaling. We propose that the fine balance between self-renewal and differentiation is homeostatically regulated by APP, which may contribute to human-specific temporal patterns of neurogenesis.

Single-cell analysis of the postnatal dorsal V-SVZ reveals a role for Bmpr1a signaling in silencing pallial germinal activity.

The ventricular-subventricular zone (V-SVZ) is the largest neurogenic region of the postnatal forebrain, containing neural stem cells (NSCs) that emerge from both the embryonic pallium and subpallium. Despite of this dual origin, glutamatergic neurogenesis declines rapidly after birth, while GABAergic neurogenesis persists throughout life. We performed single-cell RNA sequencing of the postnatal dorsal V-SVZ for unraveling the mechanisms leading to pallial lineage germinal activity silencing. We show that pallial NSCs enter a state of deep quiescence, characterized by high bone morphogenetic protein (BMP) signaling, reduced transcriptional activity and Hopx expression, while in contrast, subpallial NSCs remain primed for activation. Induction of deep quiescence is paralleled by a rapid blockade of glutamatergic neuron production and differentiation. Last, manipulation of Bmpr1a demonstrates its key role in mediating these effects. Together, our results highlight a central role of BMP signaling in synchronizing quiescence induction and blockade of neuronal differentiation to rapidly silence pallial germinal activity after birth.

Diversity within olfactory sensory derivatives revealed by the contribution of Dbx1 lineages.

In vertebrates, the embryonic olfactory epithelium contains progenitors that will give rise to distinct classes of neurons, including olfactory sensory neurons (OSNs; involved in odor detection), vomeronasal sensory neurons (VSNs; responsible for pheromone sensing), and gonadotropin-releasing hormone (GnRH) neurons that control the hypothalamic-pituitary-gonadal axis. Currently, these three neuronal lineages are usually believed to emerge from uniform pools of progenitors. Here, we found that the homeodomain transcription factor Dbx1 is expressed by neurogenic progenitors in the developing and adult mouse olfactory epithelium. We demonstrate that Dbx1 itself is dispensable for neuronal fate specification and global organization of the olfactory sensory system. Using lineage tracing, we characterize the contribution of Dbx1 lineages to OSN, VSN, and GnRH neuron populations and reveal an unexpected degree of diversity. Furthermore, we demonstrate that Dbx1-expressing progenitors remain neurogenic in the absence of the proneural gene Ascl1. Our work therefore points to the existence of distinct neurogenic programs in Dbx1-derived and other olfactory lineages.

Androgens show sex-dependent differences in myelination in immune and non-immune murine models of CNS demyelination.

Neuroprotective, anti-inflammatory, and remyelinating properties of androgens are well-characterized in demyelinated male mice and men suffering from multiple sclerosis. However, androgen effects mediated by the androgen receptor (AR), have been only poorly studied in females who make low androgen levels. Here, we show a predominant microglial AR expression in demyelinated lesions from female mice and women with multiple sclerosis, but virtually undetectable AR expression in lesions from male animals and men with multiple sclerosis. In female mice, androgens and estrogens act in a synergistic way while androgens drive microglia response towards regeneration. Transcriptomic comparisons of demyelinated mouse spinal cords indicate that, regardless of the sex, androgens up-regulate genes related to neuronal function integrity and myelin production. Depending on the sex, androgens down-regulate genes related to the immune system in females and lipid catabolism in males. Thus, androgens are required for proper myelin regeneration in females and therapeutic approaches of demyelinating diseases need to consider male-female differences.

Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation.

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves large-scale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-ß1, a key mediator of survival at this transient stage. Constitutive <i>Tns3</i> loss of function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of <i>Tns3</i> in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a twofold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-ß1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

Chromatin remodelers in oligodendroglia.

Oligodendrocytes, the myelinating cells in the vertebrate central nervous system, produce myelin sheaths to enable saltatory propagation of action potentials. The process of oligodendrocyte myelination entails a stepwise progression from precursor specification to differentiation, which is coordinated by a series of transcriptional and chromatin remodeling events. ATP-dependent chromatin remodeling enzymes, which utilize ATP as an energy source to control chromatin dynamics and regulate the accessibility of chromatin to transcriptional regulators, are critical for oligodendrocyte lineage development and regeneration. In this review, we focus on the latest insights into the spatial and temporal specificity of chromatin remodelers during oligodendrocyte development, myelinogenesis, and regeneration. We will also bring together various plausible mechanisms by which lineage specific transcriptional regulators coordinate with chromatin remodeling factors for programming genomic landscapes to specifically modulate these different processes during developmental myelination and remyelination upon injury.

Oligodendrocyte precursor survival and differentiation requires chromatin remodeling by Chd7 and Chd8.

Oligodendrocyte precursor cells (OPCs) constitute the main proliferative cells in the adult brain, and deregulation of OPC proliferation-differentiation balance results in either glioma formation or defective adaptive (re)myelination. OPC differentiation requires significant genetic reprogramming, implicating chromatin remodeling. Mounting evidence indicates that chromatin remodelers play important roles during normal development and their mutations are associated with neurodevelopmental defects, with CHD7 haploinsuficiency being the cause of CHARGE syndrome and CHD8 being one of the strongest autism spectrum disorder (ASD) high-risk-associated genes. Herein, we report on uncharacterized functions of the chromatin remodelers Chd7 and Chd8 in OPCs. Their OPC-chromatin binding profile, combined with transcriptome and chromatin accessibility analyses of Chd7-deleted OPCs, demonstrates that Chd7 protects nonproliferative OPCs from apoptosis by chromatin closing and transcriptional repression of p53 Furthermore, Chd7 controls OPC differentiation through chromatin opening and transcriptional activation of key regulators, including Sox10, Nkx2.2, and Gpr17 However, Chd7 is dispensable for oligodendrocyte stage progression, consistent with Chd8 compensatory function, as suggested by their common chromatin-binding profiles and genetic interaction. Finally, CHD7 and CHD8 bind in OPCs to a majority of ASD risk-associated genes, suggesting an implication of oligodendrocyte lineage cells in ASD neurological defects. Our results thus offer new avenues to understand and modulate the CHD7 and CHD8 functions in normal development and disease.

Dual Requirement of CHD8 for Chromatin Landscape Establishment and Histone Methyltransferase Recruitment to Promote CNS Myelination and Repair.

Disruptive mutations in chromatin remodeler CHD8 cause autism spectrum disorders, exhibiting widespread white matter abnormalities; however, the underlying mechanisms remain elusive. We show that cell-type specific Chd8 deletion in oligodendrocyte progenitors, but not in neurons, results in myelination defects, revealing a cell-intrinsic dependence on CHD8 for oligodendrocyte lineage development, myelination and post-injury remyelination. CHD8 activates expression of BRG1-associated SWI/SNF complexes that in turn activate CHD7, thus initiating a successive chromatin remodeling cascade that orchestrates oligodendrocyte lineage progression. Genomic occupancy analyses reveal that CHD8 establishes an accessible chromatin landscape, and recruits MLL/KMT2 histone methyltransferase complexes distinctively around proximal promoters to promote oligodendrocyte differentiation. Inhibition of histone demethylase activity partially rescues myelination defects of CHD8-deficient mutants. Our data indicate that CHD8 exhibits a dual function through inducing a cascade of chromatin reprogramming and recruiting H3K4 histone methyltransferases to establish oligodendrocyte identity, suggesting potential strategies of therapeutic intervention for CHD8-associated white matter defects.

Chd7 cooperates with Sox10 and regulates the onset of CNS myelination and remyelination.

Mutations in CHD7, encoding ATP-dependent chromodomain helicase DNA-binding protein 7, in CHARGE syndrome lead to multiple congenital anomalies, including craniofacial malformations, neurological dysfunction and growth delay. Mechanisms underlying the CNS phenotypes remain poorly understood. We found that Chd7 is a direct transcriptional target of oligodendrogenesis-promoting factors Olig2 and Smarca4/Brg1 and is required for proper onset of CNS myelination and remyelination. Genome-occupancy analyses in mice, coupled with transcriptome profiling, revealed that Chd7 interacted with Sox10 and targeted the enhancers of key myelinogenic genes. These analyses identified previously unknown Chd7 targets, including bone formation regulators Osterix (also known as Sp7) and Creb3l2, which are also critical for oligodendrocyte maturation. Thus, Chd7 coordinates with Sox10 to regulate the initiation of myelinogenesis and acts as a molecular nexus of regulatory networks that account for the development of a seemingly diverse array of lineages, including oligodendrocytes and osteoblasts, pointing to previously uncharacterized Chd7 functions in white matter pathogenesis in CHARGE syndrome.

Vsx1 Transiently Defines an Early Intermediate V2 Interneuron Precursor Compartment in the Mouse Developing Spinal Cord.

Spinal ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities. Interneurons arise during embryonic development from distinct progenitor domains distributed orderly along the dorso-ventral axis of the neural tube. A single ventral progenitor population named p2 generates at least five V2 interneuron subsets. Whether the diversification of V2 precursors into multiple subsets occurs within the p2 progenitor domain or involves a later compartment of early-born V2 interneurons remains unsolved. Here, we provide evidence that the p2 domain produces an intermediate V2 precursor compartment characterized by the transient expression of the transcriptional repressor Vsx1. These cells display an original repertoire of cellular markers distinct from that of any V2 interneuron population. They have exited the cell cycle but have not initiated neuronal differentiation. They coexpress Vsx1 and Foxn4, suggesting that they can generate the known V2 interneuron populations as well as possible additional V2 subsets. Unlike V2 interneurons, the generation of Vsx1-positive precursors does not depend on the Notch signaling pathway but expression of Vsx1 in these cells requires Pax6. Hence, the p2 progenitor domain generates an intermediate V2 precursor compartment, characterized by the presence of the transcriptional repressor Vsx1, that contributes to V2 interneuron development.

Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis.

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

Neural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

Oligodendroglial maturation is dependent on intracellular protein shuttling.

Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches.

Ascl1 controls the number and distribution of astrocytes and oligodendrocytes in the gray matter and white matter of the spinal cord.

Glia constitute the majority of cells in the mammalian central nervous system and are crucial for neurological function. However, there is an incomplete understanding of the molecular control of glial cell development. We find that the transcription factor Ascl1 (Mash1), which is best known for its role in neurogenesis, also functions in both astrocyte and oligodendrocyte lineages arising in the mouse spinal cord at late embryonic stages. Clonal fate mapping in vivo reveals heterogeneity in Ascl1-expressing glial progenitors and shows that Ascl1 defines cells that are restricted to either gray matter (GM) or white matter (WM) as astrocytes or oligodendrocytes. Conditional deletion of Ascl1 post-neurogenesis shows that Ascl1 is required during oligodendrogenesis for generating the correct numbers of WM but not GM oligodendrocyte precursor cells, whereas during astrocytogenesis Ascl1 functions in balancing the number of dorsal GM protoplasmic astrocytes with dorsal WM fibrous astrocytes. Thus, in addition to its function in neurogenesis, Ascl1 marks glial progenitors and controls the number and distribution of astrocytes and oligodendrocytes in the GM and WM of the spinal cord.

Development of the prethalamus is crucial for thalamocortical projection formation and is regulated by Olig2.

Thalamocortical axons (TCAs) pass through the prethalamus in the first step of their neural circuit formation. Although it has been supposed that the prethalamus is an intermediate target for thalamocortical projection formation, much less is known about the molecular mechanisms of this targeting. Here, we demonstrated the functional implications of the prethalamus in the formation of this neural circuit. We show that Olig2 transcription factor, which is expressed in the ventricular zone (VZ) of prosomere 3, regulates prethalamus formation, and loss of Olig2 results in reduced prethalamus size in early development, which is accompanied by expansion of the thalamic eminence (TE). Extension of TCAs is disorganized in the Olig2-KO dorsal thalamus, and initial elongation of TCAs is retarded in the Olig2-KO forebrain. Microarray analysis demonstrated upregulation of several axon guidance molecules, including Epha3 and Epha5, in the Olig2-KO basal forebrain. In situ hybridization showed that the prethalamus in the wild type excluded the expression of Epha3 and Epha5, whereas loss of Olig2 resulted in reduction of this Ephas-negative area and the corresponding expansion of the Ephas-positive TE. Dissociated cultures of thalamic progenitor cells demonstrated that substrate-bound EphA3 suppresses neurite extension from dorsal thalamic neurons. These results indicate that Olig2 is involved in correct formation of the prethalamus, which leads to exclusion of the EphA3-expressing region and is crucial for proper TCA formation. Our observation is the first report showing the molecular mechanisms underlying how the prethalamus acts on initial thalamocortical projection formation.

Organelle and cellular abnormalities associated with hippocampal heterotopia in neonatal doublecortin knockout mice.

Heterotopic or aberrantly positioned cortical neurons are associated with epilepsy and intellectual disability. Various mouse models exist with forms of heterotopia, but the composition and state of cells developing in heterotopic bands has been little studied. Dcx knockout (KO) mice show hippocampal CA3 pyramidal cell lamination abnormalities, appearing from the age of E17.5, and mice suffer from spontaneous epilepsy. The Dcx KO CA3 region is organized in two distinct pyramidal cell layers, resembling a heterotopic situation, and exhibits hyperexcitability. Here, we characterized the abnormally organized cells in postnatal mouse brains. Electron microscopy confirmed that the Dcx KO CA3 layers at postnatal day (P) 0 are distinct and separated by an intermediate layer devoid of neuronal somata. We found that organization and cytoplasm content of pyramidal neurons in each layer were altered compared to wild type (WT) cells. Less regular nuclei and differences in mitochondria and Golgi apparatuses were identified. Each Dcx KO CA3 layer at P0 contained pyramidal neurons but also other closely apposed cells, displaying different morphologies. Quantitative PCR and immunodetections revealed increased numbers of oligodendrocyte precursor cells (OPCs) and interneurons in close proximity to Dcx KO pyramidal cells. Immunohistochemistry experiments also showed that caspase-3 dependent cell death was increased in the CA1 and CA3 regions of Dcx KO hippocampi at P2. Thus, unsuspected ultrastructural abnormalities and cellular heterogeneity may lead to abnormal neuronal function and survival in this model, which together may contribute to the development of hyperexcitability.

Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination.

Oligodendrocytes are the myelin-forming cells of the CNS. They differentiate from oligodendrocyte precursor cells (OPCs) that are produced from progenitors throughout life but more actively during the neonatal period and in response to demyelinating insults. An accurate regulation of oligodendrogenesis is required to generate oligodendrocytes during these developmental or repair processes. We hypothesized that this regulation implicates transcription factors, which are expressed by OPCs and/or their progenitors. Ascl1/Mash1 is a proneural transcription factor previously implicated in embryonic oligodendrogenesis and operating in genetic interaction with Olig2, an essential transcriptional regulator in oligodendrocyte development. Herein, we have investigated the contribution of Ascl1 to oligodendrocyte development and remyelination in the postnatal cortex. During the neonatal period, Ascl1 expression was detected in progenitors of the cortical subventricular zone and in cortical OPCs. Different genetic approaches to delete Ascl1 in cortical progenitors or OPCs reduced neonatal oligodendrogenesis, showing that Ascl1 positively regulated both OPC specification from subventricular zone progenitors as well as the balance between OPC differentiation and proliferation. Examination of remyelination processes, both in the mouse model for focal demyelination of the corpus callosum and in multiple sclerosis lesions in humans, indicated that Ascl1 activity was upregulated along with increased oligodendrogenesis observed in remyelinating lesions. Additional genetic evidence indicated that remyelinating oligodendrocytes derived from Ascl1(+) progenitors/OPCs and that Ascl1 was required for proper remyelination. Together, our results show that Ascl1 function modulates multiple steps of OPC development in the postnatal brain and in response to demyelinating insults.

Opposing roles for Hoxa2 and Hoxb2 in hindbrain oligodendrocyte patterning.

Oligodendrocytes are the myelin-forming cells of the vertebrate CNS. Little is known about the molecular control of region-specific oligodendrocyte development. Here, we show that oligodendrogenesis in the mouse rostral hindbrain, which is organized in a metameric series of rhombomere-derived (rd) territories, follows a rhombomere-specific pattern, with extensive production of oligodendrocytes in the pontine territory (r4d) and delayed and reduced oligodendrocyte production in the prepontine region (r2d, r3d). We demonstrate that segmental organization of oligodendrocytes is controlled by Hox genes, namely Hoxa2 and Hoxb2. Specifically, Hoxa2 loss of function induced a dorsoventral enlargement of the Olig2/Nkx2.2-expressing oligodendrocyte progenitor domain, whereas conditional Hoxa2 overexpression in the Olig2(+) domain inhibited oligodendrogenesis throughout the brain. In contrast, Hoxb2 deletion resulted in a reduction of the pontine oligodendrogenic domain. Compound Hoxa2(-/-)/Hoxb2(-/-) mutant mice displayed the phenotype of Hoxb2(-/-) mutants in territories coexpressing Hoxa2 and Hoxb2 (rd3, rd4), indicating that Hoxb2 antagonizes Hoxa2 during rostral hindbrain oligodendrogenesis. This study provides the first in vivo evidence that Hox genes determine oligodendrocyte regional identity in the mammalian brain.

A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets.

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.