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Mechanisms that regulate the number of neurons during mouse neocortical development
Abstract
Cortical development progresses through an early phase of progenitor expansion, a middle phase of neurogenesis, and a final phase of gliogenesis. During the middle phase, the neurogenic phase, the neocortical primordium balances the production of neurons against the maintenance of neural precursor cells (NPCs). The final number of neurons is determined by the duration of the neurogenic phase, the rate of NPC division, and the mode of NPC division, that is, whether a division gives rise to two NPCs, one NPC and one cell committed to the neuronal lineage, or two committed cells. We discuss here recent advances in understanding these key aspects that are fundamental for normal brain development.
... Indeed, during the initial expansion phase, NPCs exclusively undergo proliferative symmetric divisions to generate a sufficiently large pool of themselves. During the following neurogenic phase, NPCs go through irreversible differentiative asymmetric divisions to produce self-renewing NPCs as well as differentiated intermediate progenitors or specific subtypes of neurons in a developmental stage-specific manner (Miyata et al., 2010;Taverna et al., 2014). During development of the mouse telencephalon, the expansion and neurogenic phases correspond to approximately embryonic days (E) 8.5-E10.5 and E10.5-E17.5, respectively (Takahashi et al., 1999). ...
... In mouse NPCs, several cellular characteristics differ between these phases, including the location of mitosis within the tissue (Haubensak et al., 2004;Miyata et al., 2004), the proliferative capacity (Calegari, 2005) and the cleavage plane orientation to the ventricular surface (Postiglione et al., 2011;Shitamukai et al., 2011). Therefore, these complex cellular changes need to be carefully orchestrated during the transition from the expansion to the neurogenic phase (Miyata et al., 2010;Taverna et al., 2014). This phase transition has been demonstrated to depend on Notch (Hatakeyama et al., 2004) and FGF (Sahara and O'Leary, 2009) signaling, as well as all-trans retinoic acid secreted from the meninges (Siegenthaler et al., 2009). ...
... During brain development, the final number of neurons is highly dependent on the size of the NPC pool at the onset of neurogenesis. Thus, the timing of the transition from the proliferative to the neurogenic division phase is tightly controlled in NPCs (Miyata et al., 2010;Rakic, 1995;Taverna et al., 2014). Here, we demonstrated that PAR3 is required for this appropriate transition by restricting proliferative NPC divisions during early telencephalic development, especially in the ventral region. ...
During brain development, neural precursor cells (NPCs) expand initially, and then switch to generating stage-specific neurons while maintaining self-renewal ability. Because the NPC pool at the onset of neurogenesis critically affects the final number of each type of neuron, tight regulation is necessary for the transitional timing from the expansion to neurogenic phase in these cells. However, the molecular mechanisms underlying this transition are poorly understood. Here, we report that the telencephalon-specific loss of PAR3 before the start of neurogenesis leads to increased NPC proliferation in dispense of neurogenesis, resulting in disorganized tissue architecture. These NPCs demonstrate hyperactivation of Hedgehog signaling in a Smoothened-dependent manner, as well as defects in primary cilia. Furthermore, loss of PAR3 enhanced ligand-independent ciliary accumulation of Smoothened and an inhibitor of Smoothened ameliorated the hyperproliferation of NPCs in the telencephalon. Thus, these findings support the idea that PAR3 has a critical role in the transition of NPCs from the expansion to neurogenic phase by restricting Hedgehog signaling through the establishment of ciliary integrity.
... The combination of the expression level of these different transcription factors provide positional information for the cortical progenitors to form different cortical areas (Arai & Pierani, 2014;O'Leary & Sahara, 2008). Coincident with this process, distinct cortical layers (2)(3)(4)(5)(6), and the anatomically and functionally distinct areas seen in the adult, differentiate from the CP. Genes that are differentially expressed across the cortex are often expressed in different patterns in different layers, suggesting that area-specific regulation of such genes is modulated by layer-specific properties, and questions the definition of area identity. ...
... During the early developmental stages, cortical progenitors undergo symmetric divisions, resulting in the proliferation of progenitors and thereby allowing expansion of the developing cortex. Soon after, cortical progenitors start generating distinct types of neurons through asymmetric differentiative divisions (1)(2)(3)(4)(5). The precise timing of the switch from proliferative division to differentiative division is crucial to determining the number of cortical neurons, and thus the cortical size. ...
A tight control of the organization of the cerebral cortex is vital for most species. Understanding the regulatory mechanisms supporting these processes is an important endeavor in developmental biology. We focused on the processes taking place during neurogenesis of the cerebral cortex, and took a multi-disciplinary approach combining biological experiments and mathematical models. We start with a model describing the probability of progenitor divisions sequence as a function of time during the neurogenesis. It is parsimonious, but sufficient to explain and draw predictions on the phenotype observed in Lhx2 conditional knock-out mutant that precocious neurogenesis affected cortical surface and thickness. The second topic we studied is how the brain compensates cell death to maintain homeostasis during development. In two mutant mouse models with neuronal death between embryonic days 11 to 14, different compensation phenotypes are observed. Here, we develop a unified mathematical model that reconciles those two opposite observations. This model is based on two fundamental compensation mechanisms: 1) an increase in the probability and maximal number of intermediate progenitor proliferative divisions; 2) a delay in the switching time between upper- and deep-layer neurons generation by a maximum of 24h. The third topic we studied is the role of extracellular Pax6 on neurogenesis. Pax6 is one of the master regulators of neuronal progenitor proliferative division and differentiation. With its homeodomain, it can transfer between cells and exert non-cell autonomous activities. We showed that blocking extracellular Pax6 induces Cajal-Retzius neurons generation ectopically.
... Defects in these processes lead to brain disorders, including autosomal recessive primary microcephaly (MCPH) [5][6][7][8]. During cortical development, the balance between symmetric and asymmetric cell division of NPCs determines the size of the NPC pool for ongoing neurogenesis and ultimately brain size [9][10][11][12][13][14][15][16]. Disturbance in symmetric cell division leads to a reduction in the NPC pool and a falloff of neuron production [10,14,[16][17][18][19]. ...
... During cortical development, the balance between symmetric and asymmetric cell division of NPCs determines the size of the NPC pool for ongoing neurogenesis and ultimately brain size [9][10][11][12][13][14][15][16]. Disturbance in symmetric cell division leads to a reduction in the NPC pool and a falloff of neuron production [10,14,[16][17][18][19]. ...
Mutations of WD repeat domain 62 (WDR62) lead to autosomal recessive primary microcephaly (MCPH), and down-regulation of WDR62 expression causes the loss of neural progenitor cells (NPCs). However, how WDR62 is regulated and hence controls neurogenesis and brain size remains elusive. Here, we demonstrate that mitogen-activated protein kinase kinase kinase 3 (MEKK3) forms a complex with WDR62 to promote c-Jun N-terminal kinase (JNK) signaling synergistically in the control of neurogenesis. The deletion of Mekk3, Wdr62, or Jnk1 resulted in phenocopied defects, including premature NPC differentiation. We further showed that WDR62 protein is positively regulated by MEKK3 and JNK1 in the developing brain and that the defects of wdr62 deficiency can be rescued by the transgenic expression of JNK1. Meanwhile, WDR62 is also negatively regulated by T1053 phosphorylation, leading to the recruitment of F-box and WD repeat domain-containing protein 7 (FBW7) and proteasomal degradation. Our findings demonstrate that the coordinated reciprocal and bidirectional regulation among MEKK3, FBW7, WDR62, and JNK1, is required for fine-tuned JNK signaling for the control of balanced NPC self-renewal and differentiation during cortical development.
... Dmrta2 | Hes1 | cell cycle | transcription factor | neurogenesis B alancing neural progenitor cell (NPC) self-renewal and neuronal differentiation is essential for generating cells in correct numbers and diverse types during brain development (1,2). As such, cortical neurogenesis is tightly regulated by a complex array of transcription factors that work in concert to coordinate NPC maintenance and differentiation. ...
... Significant disruptions to this delicate regulatory network can result in severe developmental defects due to altered neuronal production (1,2). One such disorder is microlissencephaly, a rare genetic-linked group of neurodevelopmental malformations characterized by the absence of sulci and gyri of the cerebral cortex and an accompanying reduction in cortical size and volume. ...
Significance
Maintaining an intricate balance between continued progenitor proliferation and cell cycle exit/differentiation is pivotal for proper brain development. Disruption of this delicate process can lead to brain malformations, such as microlissencephaly. In this paper, we identify Dmrta2 (doublesex- and mab-3–related transcription factor a2, also known as Dmrt5) as an important transcription factor that helps regulate the fine tuning between cell cycle progression and neuronal differentiation. Mechanistically, this function of Dmrta2 involves direct transcriptional regulation of a known repressor of neurogenesis Hes1. Our findings thus add Dmrta2 to the complex regulatory machinery controlling cortical NPC maintenance, and provide an explanation for the microlissencephaly caused by Dmrta2 deficiency in model organisms and humans.
... As development proceeds, RG cells divide asymmetrically to produce post-mitotic neurons or intermediate neuronal progenitor (INP) cells. INP cells localise in the subventricular zone (SVZ), divide a few times (1-2), and differentiate into neurons (Götz and Huttner, 2005; Miyata et al., 2010; Noctor et al., 2004). Newly born neurons migrate through the intermediate zone (IZ) and eventually give rise, in an inside-out manner, to five cortical layers known as layers II to VI (with layer I being essentially composed of axons and dendritic tufts) (Rakic, 1974; Sidman and Rakic, 1973 ). ...
... They originate at three focal points in the embryonic telencephalon (the cortical hem, the septum and the ventral pallium) and spread tangentially to cover the cortical plate, thus forming layer I (Borrell and Marin, 2006). A tight control of all these coordinated steps is required during early corticogenesis and involves specific transcription factors, as well as signalling molecules (for review, see Miyata et al., 2010). One candidate signal is retinoic acid (RA), a key regulator of several processes during development and organogenesis (Rhinn and Dollé, 2012 for a review). ...
Retinoic acid (RA) is a diffusible molecule involved in early forebrain patterning. Its later production in the meninges by the retinaldehyde dehydrogenase RALDH2 coincides with the time of cortical neuron generation. A function of RA in this process has not been adressed directly as Raldh2(-/-) mouse mutants are embryonic lethal. Here we used a conditional genetic strategy to inactivate Raldh2 just prior to its onset of expression in the developing meninges. This inactivation does not affect the formation of the cortical progenitor populations, their rate of division, or timing of differentiation. However, migration of late-born cortical neurons is delayed, with neurons stalling in the intermediate zone and exhibiting an abnormal multipolar morphology. This suggests that RA controls the multipolar-to-bipolar transition which occurs in the intermediate zone and allows neurons to start locomotion in the cortical plate. Our work also shows a role for RA in cortical lamination, as deep layers are expanded and a subset of layer IV neurons are not formed in the Raldh2-ablated mutants. These data demonstrate that meninges are a source of extrinsic signals important for cortical development.
... B) New engineering organoids containing microglia have been developed last year to study novel mechanisms explaining COVID-19 neurological anomalies. (Rubenstein and Rakic, 1999;Miyata et al., 2010;Kwan et al., 2012;Greig et al., 2013;Wamsley et al., 2018;Di Bella et al., 2021;Bonnefont and Vanderhaeghen, 2021) in a precise spatial-temporal manner. Within this choreographic arrangement, a single out-of-tune event in time or space may represent the inception of a neurodevelopmental pathological condition. ...
Several advances in the field of neurodevelopmental diseases (NDDs) have been reported by 2022. Of course, NDDs comprise a diverse group of disorders, most of which with different aetiologies. However, owing to the development and consolidation of technological approaches, such as proteomics and RNA-sequencing, and to the improvement of brain organoids along with the introduction of artificial intelligence (AI) for biodata analysis, in 2022 new aetiological mechanisms for some NDDs have been proposed. Here, we present hints of some of these findings. For instance, centrioles regulate neuronal migration and could be behind the aetiology of periventricular heterotopia; also, the accumulation of misfolded proteins could explain the neurological effects in COVID-19 patients; and, autism spectrum disorders (ASD) could be the expression of altered cortical arealization. We also cover other interesting aspects as the description of a new NDD characterized by deregulation of genes involved in stress granule (SG) assemblies, or the description of a newly discovered neural progenitor that explains the different phenotypes of tumours and cortical tubers in tuberous sclerosis complex (TSC) disease; and how it is possible to decipher the aetiology of sudden unexplained death in childhood (SUDC) or improve the diagnosis of cortical malformations using formalin-fixed paraffin-embedded samples.
... The concept of neurogenetic potential has been reported previously. It is illustrated as a pool of immature neurons generated in surplus but selectively eliminated by apoptosis when they are no longer recruited to the functional neural circuits in the cerebral cortex (Biebl et al., 2000;Blaschke et al., 1996;Kempermann et al., 2003;Miyata et al., 2010). Appropriate functional stimulations recruit more new neurons. ...
Neurogenesis in the subventricular zone (SVZ), subgranular zone (SGZ), and cerebral cortex is now a familiar event to confirm by cerebral arterial ischemia in rat models. However, it remains unclear whether cerebral venous ischemia (CVI) alone causes neurogenesis, and where that neurogenesis occurs. After creating CVI rat models via a two-vein occlusion (2-VO) method, neurogenesis was immunohistochemically evaluated by double-labeling 5-bromo-2’-deoxyuridine (BrdU)-positive cells with neuronal nuclei (NeuN) or doublecortin (DCX) antibody. Fifty Wistar rats were divided into two major groups (BrdU-NeuN and BrdU-DCX) and then separated into two subgroups (2-VO or sham). The total number of double-positive cells expressed inside a predefined region of interest (ROI) covering the ischemic area was compared between the two subgroups. Then, we divided the ROI into six sections to evaluate and compare the distribution of double-positive cells generated in each section between the two subgroups. The 2-VO subgroup presented more double-positive cells than the sham group in both BrdU-NeuN and BrdU-DCX groups, while the BrdU-DCX+2-VO group showed a characteristic distribution of double-positive cells in ROI 2 and ROI 3, suggesting areas of the ischemic core and penumbra, with a significant difference compared to the BrdU-DCX+sham group. This study demonstrates that CVI has the potential to induce endogenous neurogenesis, with significant numbers of both newly generated neurons and precursors observed in the ischemic area. The distribution of these cells suggests that the cortex could be the main origin of neurogenesis after cortical CVI.
... The temporal and spatial specifications of NPs are essential for CNS histogenesis [89]. Temporal changes in NP differentiation are driven by a combination of intrinsic cellular properties and extracellular signals from the environment of the developing brain [90]. Neural progenitors express GABA A receptors in the developing cortex [91,92]. ...
Mammals can obtain taurine from food and synthesize it from sulfur-containing amino acids. Mammalian fetuses and infants have little ability to synthesize taurine. Therefore, they are dependent on taurine given from mothers either via the placenta or via breast milk. Many lines of evidence demonstrate that maternally derived taurine is essential for offspring development, shaping various traits in adults. Various environmental factors, including maternal obesity, preeclampsia, and undernutrition, can affect the efficacy of taurine transfer via either the placenta or breast milk. Thus, maternally derived taurine during the perinatal period can influence the offspring’s development and even determine health and disease later in life. In this review, I will discuss the biological function of taurine during development and the regulatory mechanisms of taurine transport from mother to offspring. I also refer to the possible environmental factors affecting taurine functions in mother-offspring bonding during perinatal periods. The possible functions of taurine as a determinant of gut microbiota and in the context of the Developmental Origins of Health and Disease (DOHaD) hypothesis will also be discussed.
... During cortical development, the final neural output is determined by the duration of the neurogenic phase, the rate of neural progenitor division, the mode of RGC division, and the cell cycle duration (Miyata et al. 2010;Franco and Muller 2013;Stouffer et al. 2016). Short cell cycles, especially a shortened G1 phase, correlate with a Embryonic brains were subjected to IUE at E14.5 and analyzed at E16.5. ...
During cortical development, the balance between progenitor self-renewal and neurogenesis is critical for determining the size/morphology of the cortex. A fundamental feature of the developing cortex is an increase in the length of G1 phase in RGCs over the course of neurogenesis, which is a key determinant of progenitor fate choice. How the G1 length is temporally regulated remains unclear. Here, Pdk1, a member of the AGC kinase family, was conditionally disrupted by crossing an Emx1-Cre mouse line with a Pdk1fl/fl line. The loss of Pdk1 led to a shorter cell cycle accompanied by increased RGC proliferation specifically at late rather than early/middle neurogenic stages, which was attributed to impaired lengthening of G1 phase. Coincidently, apical-to-basal interkinetic nuclear migration was accelerated in Pdk1 cKO cortices. Consequently, we detected an increased neuronal output at P0. We further showed the significant upregulation of the cell cycle regulator cyclin D1 and its activator Myc in the cKO cortices relative to those of control animals. Overall, we have identified a novel role for PDK1 in cortical neurogenesis. PDK1 functions as an upstream regulator of the Myc-cyclin D1 pathway to control the lengthening of G1 phase and the balance between RGC proliferation and differentiation.
... In contrast to NPs, RG show several astroglial properties with the expression of various molecules that are characteristic of astrocytes-such as the astrocyte-specific glutamate transporter (GLAST), the Ca 2+ binding protein S100β, glial fibrillary acidic protein (GFAP), vimentin, and brain-lipid-binding protein (BLBP). After turning into RG, NPs start to exhibit asymmetric division to differentiate into the glutamatergic excitatory neurons in the developing cortex (Gotz and Huttner 2005;Miyata et al. 2010;Agirman et al. 2017). After the onset of neurogenesis, dividing progenitors start to appear at the basal border of the VZ. ...
Temporal specification of the neural progenitors (NPs) producing excitatory glutamatergic neurons is essential for histogenesis of the cerebral cortex. Neuroepithelial cells, the primary NPs, transit to radial glia (RG). To coincide with the transition, NPs start to differentiate into neurons, undergoing a switch from symmetric to asymmetric cell division. After the onset of neurogenesis, NPs produce layer-specific neurons in a defined order with precise timing. Here, we show that GABAA receptors (GABAARs) and taurine are involved in this regulatory mechanism. Foetal exposure to GABAAR-antagonists suppressed the transition to RG, switch to asymmetric division, and differentiation into upper-layer neurons. Foetal exposure to GABAAR-agonists caused the opposite effects. Mammalian foetuses are dependent on taurine derived from the mothers. GABA and taurine function as endogenous ligands for GABAARs. Ca2+ imaging showed that NPs principally responded to taurine but not GABA before E13. The histological phenotypes of the taurine transporter knockout mice resembled those of the mice foetally exposed to GABAAR-antagonists. Foetal exposure to GABAAR-modulators resulted in considerable alterations in offspring behavior like core symptoms of autism. These results show that taurine regulates the temporal specification of NPs and that disrupting the taurine-receptor interaction possibly leads to neurodevelopmental disorders.
... The cerebral cortex of mammalian brain is organized into layers of specialized neuronal subtypes by orchestrated stem cell maintenance, expansion, fate commitment, and differentiation (Farkas and Huttner, 2008;Miyata et al., 2010;Taverna et al., 2014). In the developing cortex, radial glial cells (RGCs) act as neural stem/progenitor cells and give rise to neurons in a systematic fashion (Taverna et al., 2014). ...
Neurogenesis in the developing neocortex relies on extensive mitosis of radial glial cells (RGCs) in the apical surface. The nuclear migration of epithelial-like RGCs is fundamentally important for proper mitosis, but how the apical processes of RGCs are anchored to ensure the nucleokinetic behavior of RGCs remains unclear. Here we find that Talpid3, related to Joubert syndrome, is localized to the mother centriole of RGCs and is required for their apical mitosis. Genetic silencing of Talpid3 causes abnormal RGC delamination and thereby impairs their interkinetic nuclear migration in both cell-autonomous and non-autonomous manners. Further analyses reveal that Talpid3 associates with Ninein to regulate microtubule organization and maintain the integrity of adherens junctions to anchor RGCs. Moreover, genetic ablation of Talpid3 results in synchronized, ectopic mitosis of neural progenitors and dysregulated neurogenesis. Our study provides an intriguing perspective for the non-ciliogenic role of centriolar proteins in mediating cortical neurogenesis.
... The ECM plays many roles during neural development, from the formation of a meshwork for structural support, to the activation of signaling pathways that stimulate progenitor proliferation, either directly or indirectly (Barros et al., 2011). Prior to the onset of neurogenesis, NECs in the cortical primordium augment their number by self-amplification via symmetric divisions (Miyata et al., 2010;Fernandez et al., 2016). Already at that early stage, the ECM provides the microenvironment necessary to modulate the behavior of NECs (Perris and Perissinotto, 2000;Zimmermann and Dours-Zimmermann, 2008). ...
The evolution of the mammalian cerebral cortex leading to humans involved a remarkable sophistication of developmental mechanisms. Specific adaptations of progenitor cell proliferation and neuronal migration mechanisms have been proposed to play major roles in this evolution of neocortical development. One of the central elements influencing neocortex development is the extracellular matrix (ECM). The ECM provides both a structural framework during tissue formation and to present signaling molecules to cells, which directly influences cell behavior and movement. Here we review recent advances in the understanding of the role of ECM molecules on progenitor cell proliferation and neuronal migration, and how these contribute to cerebral cortex expansion and folding. We discuss how transcriptomic studies in human, ferret and mouse identify components of ECM as being candidate key players in cortex expansion during development and evolution. Then we focus on recent functional studies showing that ECM components regulate cortical progenitor cell proliferation, neuron migration and the mechanical properties of the developing cortex. Finally, we discuss how these features differ between lissencephalic and gyrencephalic species, and how the molecular evolution of ECM components and their expression profiles may have been fundamental in the emergence and evolution of cortex folding across mammalian phylogeny.
... There are two major types of BPs: basal (or outer) radial glia (bRGs) and basal intermediate progenitors (bIPs) [2][3][4][5][9][10][11][12][13]. These BPs may undergo three types of cell division: (1) proliferative divisions (1 BP / 2 BPs, both bRGs and bIPs), (2) self-renewing divisions (1 BP / 1 BP + 1 neuron, typically bRGs), and (3) consumptive divisions (1 BP / 2 neurons, both bRGs and bIPs) [2,10,14,15]. Given the existence of two major types of BPs and of three distinct types of their cell division, a variety of lineages from NPCs to cortical neurons can be envisioned for the various mammalian systems under study, many of which have actually been observed by live imaging of neocortical tissue ex vivo [14,[16][17][18][19][20][21][22][23]. ...
A hallmark of the evolutionary expansion of the neocortex is a specific increase in the number of neurons generated for the upper neocortical layers during development. The cause underlying this increase is unknown. Here, we show that lengthening the neurogenic period during neocortical development is sufficient to specifically increase upper-layer neuron generation. Thus, embryos of mouse strains with longer gestation exhibited a longer neurogenic period and generated more upper-layer, but not more deep-layer, neurons than embryos with shorter gestation. Accordingly, long-gestation embryos showed a greater abundance of neurogenic progenitors in the subventricular zone than short-gestation embryos at late stages of cortical neurogenesis. Analysis of a mouse-rat chimeric embryo, developing inside a rat mother, pointed to factors in the rat environment that influenced the upper-layer neuron generation by the mouse progenitors. Exploring a potential maternal source of such factors, short-gestation strain mouse embryos transferred to long-gestation strain mothers exhibited an increase in the length of the neurogenic period and upper-layer neuron generation. The opposite was the case for long-gestation strain mouse embryos transferred to short-gestation strain mothers, indicating a dominant maternal influence on the length of the neurogenic period and hence upper-layer neuron generation. In summary, our study uncovers a hitherto unknown link between embryonic cortical neurogenesis and the maternal gestational environment and provides experimental evidence that lengthening the neurogenic period during neocortical development underlies a key aspect of neocortical expansion.
... There are two major types of BPs: basal (or outer) radial glia (bRGs) and basal intermediate progenitors (bIPs) [2][3][4][5][9][10][11][12][13]. These BPs may undergo three types of cell division: (1) proliferative divisions (1 BP / 2 BPs, both bRGs and bIPs), (2) self-renewing divisions (1 BP / 1 BP + 1 neuron, typically bRGs), and (3) consumptive divisions (1 BP / 2 neurons, both bRGs and bIPs) [2,10,14,15]. Given the existence of two major types of BPs and of three distinct types of their cell division, a variety of lineages from NPCs to cortical neurons can be envisioned for the various mammalian systems under study, many of which have actually been observed by live imaging of neocortical tissue ex vivo [14,[16][17][18][19][20][21][22][23]. ...
... The undifferentiated RG progenitors are maintained in an undifferentiated state for a certain period and sequentially generate multiple excitatory subtypes of neurons in an inside-out direction; the subtypes in the deeper layers are produced earlier than those in the upper layers. The temporal regulation of cortical progenitors to produce a certain subtype of neuron in a given developmental timing are increasingly understood (Desai and McConnell, 2000;Greig et al., 2013;Hirabayashi and Gotoh, 2010;Leone et al., 2008;McConnell, 1991;McConnell and Kaznowski, 1991;Miyata et al., 2010;Mizutani and Saito, 2005;Oberst et al., 2018;Okamoto et al., 2016;Vitali et al., 2018;Yoon et al., 2017). Every single RG cell in mice produces a relatively constant number of descendant neurons allocated in multiple cortical layers regardless of its tangential position ( Gao et al., 2014). ...
One of the most important questions in human evolutionary biology is how our ancestor has acquired an expanded volume of the cerebral cortex, which may have significantly impacted on improving our cognitive abilities. Recent comparative approaches have identified developmental features unique to the human or hominid cerebral cortex, not shared with other animals including conventional experimental models. In addition, genomic, transcriptomic, and epigenomic signatures associated with human- or hominid-specific processes of the cortical development are becoming identified by virtue of technical progress in the deep nucleotide sequencing. This review discusses ontogenic and phylogenetic processes of the human cerebral cortex, followed by the introduction of recent comprehensive approaches identifying molecular mechanisms potentially driving the evolutionary changes in the cortical development.
... In humans, NEs are also the founder NPC population forming the VZ between gestational weeks 4 and 5 (GW4 and GW5) (Bystron, Blakemore, & Rakic, 2008). The main function of NEs is the amplification of the progenitor pool by symmetric proliferating divisions, which generate two daughter NEs in each round of division (Miyata, Kawaguchi, Kawaguchi, & Gotoh, 2010). The original number of NEs predetermines the size of the cortex since they set the original pool of progenitor cells. ...
The brain is one of the most complex organs, responsible for the advanced intellectual and cognitive ability of humans. Although primates are to some extent capable of performing cognitive tasks, their abilities are less evolved. One of the reasons for this is the vast differences in the brain of humans compared to other mammals, in terms of shape, size and complexity. Such differences make the study of human brain development fascinating. Interestingly, the cerebral cortex is by far the most complex brain region resulting from its selective evolution within mammals over millions of years. Unraveling the molecular and cellular mechanisms regulating brain development, as well as the evolutionary differences seen across species and the need to understand human brain disorders, are some of the reasons why scientists are interested in improving their current knowledge on human corticogenesis. Toward this end, several animal models including primates have been used, however, these models are limited in their extent to recapitulate human‐specific features. Recent technological achievements in the field of stem cell research, which have enabled the generation of human models of corticogenesis, called brain or cerebral organoids, are of great importance. This review focuses on the main cellular and molecular features of human corticogenesis and the use of brain organoids to study it. We will discuss the key differences between cortical development in human and nonhuman mammals, the technological applications of brain organoids and the different aspects of cortical development in normal and pathological conditions, which can be modeled using brain organoids.
This article is categorized under:
• Comparative Development and Evolution > Regulation of Organ Diversity
• Nervous System Development > Vertebrates: General Principles
... The properties of radial glia were identified due to similarities with astrocytes, which contain glycogen granules and express glial fibrillary acidic protein (GFAP). In addition, radial glia have been considered to support the migration of nascent neurons (9); however, in the previous decade, investigations have focused on the progenitor capacity of radial glia within the cerebral cortex (10,11) and cerebellum (11,12). ...
Radial glia are key neural progenitors involved in the development of the central nervous system. Tyrosine-protein phosphatase non‑receptor type 11 (Shp2) is a widely expressed intracellular enzyme with multiple cellular functions. Previous studies have revealed the critical role of Shp2 in a variety of neural cell types; however, further investigation into the function of Shp2 within radial glia is required. In the present study, a conditional knockout mouse was generated using a human glial fibrillary acidic protein (hGFAP)‑Cre driver, in which the Shp2 genes were deleted within radial glia. Loss of Shp2 within radial glia was associated with developmental retardation, postnatal lethality, reduced brain size and thinner cerebral cortices in newborn mice. Deletion of Shp2 also led to an increase in gliogenesis, a reduction in neural genesis and extracellular signal‑regulated kinase signaling within the cerebral cortex. Furthermore, glial cell defects within the cerebellum of Shp2 mutants were observed, with abnormal granular cell retention and glial cell alignment in the external granular layer. In addition, Shp2 mutants exhibited impaired sensory‑motor development. The results of the present study suggested that Shp2 may have an important role within radial glia, and regulate cerebral cortical and cerebellar development in newborn mice.
... Par exemple, chez l'homme, la neurogenèse débute quelques jours plus tard par rapport au singe. Ceci permet 3 à 4 divisions supplémentaires et, in fine, une expansion de la surface cortical 8 à 16 fois plus importante (Miyata et al. 2010). ...
La mutation à l’origine de la maladie de Huntington (MH) correspond à une expansion anormale de glutamines sur la protéine huntingtine (HTT). La MH est caractérisée par des symptômes moteurs et cognitifs mais également des troubles psychiatriques tels que l’anxiété et la dépression.Au cours de ma thèse, j’ai montré que la HTT module le statut anxio-dépressif de la souris via ses phosphorylations aux sérines 1181/1201. En effet, l’ablation des phosphorylations sur la HTT endogène améliore significativement le phénotype anxio-dépressif de la souris. Chez la souris, cette modulation dépend d’une augmentation de la maturation et de la survie des nouveaux neurones dans l’hippocampe. En effet, l’irradiation focale de l’hippocampe, dans un contexte où les phosphorylations sont absentes, supprime la neurogenèse et la réduction du statut anxio-dépressif observée en l’absence de phosphorylations. Au niveau moléculaire, la HTT non phosphorylée accroît l’association des moteurs moléculaires et des vésicules de BDNF sur les microtubules, ce qui augmente les dynamiques et la libération du BDNF. Ceci active la voie de signalisation MAPK/CREB dans l’hippocampe, cette voie pouvant ainsi stimuler la neurogenèse.J’ai ensuite étudié le rôle de ces phosphorylations dans un contexte MH et j’ai démontré l’effet anxiolytique/antidépresseur de l’absence de ces phosphorylations.J’ai également montré le rôle de ces phosphorylations de la HTT au cours du développement du cortex embryonnaire.Les résultats obtenus au cours de ma thèse suggèrent que les mécanismes fondamentaux de neurogenèse sont régulés par la HTT et ses phosphorylations. De plus, ils identifient une nouvelle voie de modulation de l’anxiété/dépression faisant intervenir la HTT.
... Undifferentiated neural progenitor cells (NPCs) are central to the production of a variety of cells in developing and mature brains [1][2][3][4][5][6][7][8][9]. During the early embryonic period before neurons emerge, the nuclei/somata of NPCs are diffusely distributed throughout the initial wall of brain primordia [also called the neuroepithelium (NE)]. ...
Spatiotemporally ordered production of cells is essential for brain development. Normally, most undifferentiated neural progenitor cells (NPCs) face the apical (ventricular) surface of embryonic brain walls. Pathological detachment of NPCs from the apical surface and their invasion of outer neuronal territories, i.e., formation of NPC heterotopias, can disrupt the overall structure of the brain. Although NPC heterotopias have previously been observed in a variety of experimental contexts, the underlying mechanisms remain largely unknown. Yes-associated protein 1 (Yap1) and the TEA domain (Tead) proteins, which act downstream of Hippo signaling, enhance the stem-like characteristics of NPCs. Elevated expression of Yap1 or Tead in the neural tube (future spinal cord) induces massive NPC heterotopias, but Yap/Tead-induced expansion of NPCs in the developing brain has not been previously reported to produce NPC heterotopias. To determine whether NPC heterotopias occur in a regionally characteristic manner, we introduced the Yap1-S112A or Tead-VP16 into NPCs of the telencephalon and diencephalon, two neighboring but distinct forebrain regions, of embryonic day 10 mice by in utero electroporation, and compared NPC heterotopia formation. Although NPCs in both regions exhibited enhanced stem-like behaviors, heterotopias were larger and more frequent in the diencephalon than in the telencephalon. This result, the first example of Yap/Tead-induced NPC heterotopia in the forebrain, reveals that Yap/Tead-induced NPC heterotopia is not specific to the neural tube, and also suggests that this phenomenon depends on regional factors such as the three-dimensional geometry and assembly of these cells.
... Similarly, subtle spindle deviations can also influence the inheritance of the basal process (Shitamukai et al., 2011, see also Kosodo et al., 2008). The proper regulation of these symmetry changes is thought to be an important determinant in neocortical neurogenesis (Götz and Huttner, 2005;Kriegstein and Alvarez-Buylla, 2009;Miyata et al., 2010), and its perturbation can lead to neurodevelopmental and neurodegenerative disorders (Feng and Walsh, 2004;Fish et al., 2006;Yingling et al., 2008;Gauthier-Fisher et al., 2009;Godin et al., 2010;Lizarraga et al., 2010). ...
Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis.
... In the developing telencephalon, a large number of neurons originate from neuroepithelial cells and migrate across telencephalic regions. This occurs at approximately between embryonic day (E) E10.5 and E12 in mice when neuroepithelial cells transform into radial glia cells that possess neural stem cells (NSCs)/progenitor cells features [1][2][3]. This process is tightly regulated at transcriptional level and abnormal gene expression lead to severe neurodevelopmental disorders such as autism spectrum disorders, seizure disorders, and intellectual disability [4][5][6]. ...
The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.
... An important signalling molecule involved in the control of the proliferation/differentiation switch of NSPCs is retinoic acid (RA) (Janesick et al., 2015). RA is required for neuronal differentiation and promotes expression of proneural and neurogenic genes, including Pax6 and N-tubulin (Gajovic et al., 1997;Janesick et al., 2013;Miyata et al., 2010;Siegenthaler et al., 2009). The importance of RA for neurogenesis and brain development is also reflected in the fact that genetic alterations of RA signalling components or prenatal exposure to RA have been associated with the development of HPE (Bendavid et al., 2009;Cohen and Shiota, 2002;Muenke and Beachy, 2000;Petryk et al., 2015;Roessler and Muenke, 2010). ...
Background:
Genetic deletion of Nosip in mice causes holoprosencephaly, however, the function of Nosip in neurogenesis is currently unknown.
Results:
We combined two vertebrate model organisms, the mouse and the South African clawed frog, Xenopus laevis, to study the function of Nosip in neurogenesis. We found, that size and cortical thickness of the developing brain of Nosip knockout mice were reduced. Accordingly, the formation of postmitotic neurons was greatly diminished, concomitant with a reduced number of apical and basal neural progenitor cells in vivo. Neurospheres derived from Nosip knockout embryos exhibited reduced growth and the differentiation capability into neurons in vitro was almost completely abolished. Mass spectrometry analysis of the neurospheres proteome revealed a reduced expression of Rbp1, a regulator of retinoic acid synthesis, when Nosip was absent. We identified the homologous nosip gene to be expressed in differentiated neurons in the developing brain of Xenopus embryos. Knockdown of Nosip in Xenopus resulted in a reduction of brain size that could be rescued by reintroducing human NOSIP mRNA. Furthermore, the expression of pro-neurogenic transcription factors was reduced and the differentiation of neuronal cells was impaired upon Nosip knockdown. In Xenopus as well as in mouse we identified reduced proliferation and increased apoptosis as underlying cause of microcephaly upon Nosip depletion. In Xenopus Nosip and Rbp1 are similarly expressed and knockdown of Nosip resulted in down regulation of Rbp1. Knockdown of Rbp1 caused a similar microcephaly phenotype as the depletion of Nosip and synergy experiments indicated that both proteins act in the same signalling pathway.
Conclusions:
Nosip is a novel factor critical for neural stem cell/progenitor self-renewal and neurogenesis during mouse and Xenopus development and functions upstream of Rbp1 during early neurogenesis.
... The ability of neural stem or progenitor cells (NSCs) to produce different types of neurons is determined by intrinsic genetic programs (Martynoga et al., 2012;Miyata et al., 2010;Shen et al., 2006). Cell fate can also be specified by signals from the local environment in the nervous system (Borello and Pierani, 2010;Guillemot and Zimmer, 2011). ...
... This shift initially takes place sporadically, and then progressively propagates into the entire brain with different timing. Cell-intrinsic programs and extrinsic environmental signals 6,7 control these alterations in the division mode of APs 1,8 . Notch signalling is essential for progenitor self-renewal in both the proliferative and the neurogenic mode 9,10 . ...
During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and-extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.
... This cyclic movement is known as interkinetic nuclear migration and is completely asynchronous between NECs, conferring the neuroepithelium a pseudostratified appearance (Sauer, 1935;Bayer & Altman, 1991;Taverna & Huttner, 2010). NECs only undergo symmetric self-amplificative divisions, whereby each division generates two daughter NECs, hence exponentially increasing their number (Miyata et al, 2010;Fig 1). ...
One of the most prominent features of the human brain is the fabulous size of the cerebral cortex and its intricate folding. Cortical folding takes place during embryonic development and is important to optimize the functional organization and wiring of the brain, as well as to allow fitting a large cortex in a limited cranial volume. Pathological alterations in size or folding of the human cortex lead to severe intellectual disability and intractable epilepsy. Hence, cortical expansion and folding are viewed as key processes in mammalian brain development and evolution, ultimately leading to increased intellectual performance and, eventually, to the emergence of human cognition. Here, we provide an overview and discuss some of the most significant advances in our understanding of cortical expansion and folding over the last decades. These include discoveries in multiple and diverse disciplines, from cellular and molecular mechanisms regulating cortical development and neurogenesis, genetic mechanisms defining the patterns of cortical folds, the biomechanics of cortical growth and buckling, lessons from human disease, and how genetic evolution steered cortical size and folding during mammalian evolution.
... The size of the progenitor pools and the balance of symmetric, proliferative, and asymmetric divisions determine the size of the cortex (Rakic 1988;Miyata et al. 2010). The expansion of the cortex in the Rap1 Emx1-KO mutant suggests an imbalance in the generation of progenitors and neurons. ...
During the development of the mammalian neocortex, the generation of neurons by neural progenitors and their migration to
the final position are closely coordinated. The highly polarized radial glial cells (RGCs) serve both as progenitor cells
to generate neurons and as support for the migration of these neurons. After their generation, neurons transiently assume
a multipolar morphology before they polarize and begin their migration along the RGCs. Here, we show that Rap1 GTPases perform
essential functions for cortical organization as master regulators of cell polarity. Conditional deletion of Rap1 GTPases
leads to a complete loss of cortical lamination. In RGCs, Rap1 GTPases are required to maintain their polarized organization.
In newborn neurons, the loss of Rap1 GTPases prevents the formation of axons and leading processes and thereby interferes
with radial migration. Taken together, the loss of RGC and neuronal polarity results in the disruption of cortical organization.
... During the early developmental stages, cortical progenitors undergo symmetric divisions, resulting in the proliferation of progenitors and thereby allowing expansion of the developing cortex. Soon after, cortical progenitors start generating distinct types of neurons through asymmetric differentiative divisions (1)(2)(3)(4)(5). The precise timing of the switch from proliferative division to differentiative division is crucial to determining the number of cortical neurons, and thus the cortical size. ...
Significance
The cerebral cortex is the most highly evolved structure in the human brain. Generating the correct number and types of neurons is crucial for brain function. We show a central role of the Lhx2 homeoprotein in this task: deleting Lhx2 in cortical progenitors leads to a temporal shift of neurogenesis initiation, resulting in a much smaller cortex with decreased numbers of neurons in all cortical layers. Further, we found that Lhx2 is required for the Wnt/β-catenin pathway to maintain progenitor proliferation. Using a parsimonious mathematical model, we demonstrated that such disruptions of neurogenesis timing are enough to explain the cortical size and thickness modifications observed. Our findings enlighten how neurogenesis timing is regulated molecularly and how it affects cortical size and organization.
... Radial glial progenitor cells first generate neurons in a defined temporal window during mid-gestation and subsequently generate GFAP+ astrocytes in late embryos. The neurogenesis to gliogenesis switch is highly reproducible, and intrinsic epigenetic changes that occur in the radial glial progenitor cells after completion of neurogenesis renders them responsive to signals that lead to repression of neurogenic genes and derepression of astrogliogenic genes (Miyata et al., 2010). ...
NG2 cells represent a fourth major glial cell population in the mammalian central nervous system (CNS). They arise from discrete germinal zones in mid-gestation embryos and expand to occupy the entire CNS parenchyma. Genetic fate mapping studies have shown that oligodendrocytes and a subpopulation of ventral protoplasmic astrocytes arise from NG2 cells. This review describes recent findings on the fate and fate potential of NG2 cells under physiological and pathological conditions. We discuss age-dependent changes in the fate and fate potential of NG2 cells and possible mechanisms that could be involved in restricting their oligodendrocyte differentiation or fate plasticity.
Copyright © 2015. Published by Elsevier B.V.
... Using long-term time-lapse microscopy with these markers, we provide direct evidence that GCPs increase the progenitor pool by proliferative symmetric division and then generate granule cells by terminal symmetric division. This stands in contrast to the neurogenesis in the cerebral cortex, in which self-renewing asymmetric divisions are the significant contributors to neuron generation (Miyata et al., 2010). ...
Neurons in the central nervous system (CNS) are generated by symmetric and asymmetric cell division of neural stem cells and their derivative progenitor cells. Cerebellar granule cells are the most abundant neurons in the CNS, and are generated by intensive cell division of granule cell precursors (GCPs) during postnatal development. Dysregulation of GCP cell cycle is causal for some subtypes of medulloblastoma. However, the details and mechanisms underlying neurogenesis from GCPs are not well understood.
Using long-term live-cell imaging of proliferating GCPs transfected with a fluorescent newborn-granule cell marker, we found that GCPs underwent predominantly symmetric divisions, generating two GCPs or two neurons, while asymmetric divisions generating a GCP and a neuron were only occasionally observed, in both dissociated culture and within tissues of isolated cerebellar lobules. We found no significant difference in cell cycle length between proliferative and neurogenic divisions, or any consistent changes in cell cycle length during repeated proliferative division.
Unlike neural stem cells in the cerebral cortex and spinal cord, which generate many neurons by repeated asymmetric division, cerebellar GCPs produce neurons predominantly by terminal symmetric division. These results indicate diverse mechanisms of neurogenesis in the mammalian brain. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
... One possibility is that the progressive lengthening of the progenitor cell cycle that occurs during development contributes to the increase in Neurog1 expression in mitotic progenitors. Cell cycle lengthening regulates the transition from progenitor maintenance to differentiation in the CNS [31] and cell cycle regulators have been shown to modulate Neurog2 activity [32]. Thus, changes in cell cycle protein dynamics could trigger changes in Neurog1 expression timing in later generated progenitors. ...
Neurog1 is a pro-neural basic helix-loop-helix (bHLH) transcription factor expressed in progenitor cells located in the ventricular zone and subsequently the presumptive white matter tracts of the developing mouse cerebellum. We used genetic inducible fate mapping (GIFM) with a transgenic Neurog1-CreER allele to characterize the contributions of Neurog1 lineages to cerebellar circuit formation in mice. GIFM reveals Neurog1-expressing progenitors are fate-mapped to become Purkinje cells and all GABAergic interneuron cell types of the cerebellar cortex but not glia. The spatiotemporal sequence of GIFM is unique to each neuronal cell type. GIFM on embryonic days (E) 10.5 to E12.5 labels Purkinje cells with different medial-lateral settling patterns depending on the day of tamoxifen delivery. GIFM on E11.5 to P7 labels interneurons and the timing of tamoxifen administration correlates with the final inside-to-outside resting position of GABAergic interneurons in the cerebellar cortex. Proliferative status and long-term BrdU retention of GIFM lineages reveals Purkinje cells express Neurog1 around the time they become post-mitotic. In contrast, GIFM labels mitotic and post-mitotic interneurons. Neurog1-CreER GIFM reveals a correlation between the timing of Neurog1 expression and the spatial organization of GABAergic neurons in the cerebellar cortex with possible implications for cerebellar circuit assembly.
... Between radial glial cells are tight and adherens junctions at the apical end feet, maintained through the actions of Numb and Numbl and are required for the maintenance of radial glial cell polarity [10,11]. Radial glial cells are able to divide either symmetrically or asymmetrically (Figure 1) and have been shown to undergo proliferative symmetrical cell divisions where radial glial cells or basal progenitors are produced, symmetrical neurogenic cell divisions where two neurons are produced or asymmetric cell divisions where radial glial cells, outer radial glial cells, basal progenitors or neurons are produced in combination with each other [12,13] (Figure 1 and Table 1). ...
Glioma is a heterogeneous disease process with differential histology and treatment response. It was previously thought that the histological features of glial tumors indicated their cell of origin. However, the discovery of continuous neuro-gliogenesis in the normal adult brain and the identification of brain tumor stem cells within glioma have led to the hypothesis that these brain tumors originate from multipotent neural stem or progenitor cells, which primarily divide asymmetrically during the postnatal period. Asymmetric cell division allows these cell types to concurrently self-renew whilst also producing cells for the differentiation pathway. It has recently been shown that increased symmetrical cell division, favoring the self-renewal pathway, leads to oligodendroglioma formation from oligodendrocyte progenitor cells. In contrast, there is some evidence that asymmetric cell division maintenance in tumor stem-like cells within astrocytoma may lead to acquisition of treatment resistance. Therefore cell division mode in normal brain stem and progenitor cells may play a role in setting tumorigenic potential and the type of tumor formed. Moreover, heterogeneous tumor cell populations and their respective cell division mode may confer differential sensitivity to therapy. This review aims to shed light on the controllers of cell division mode which may be therapeutically targeted to prevent glioma formation and improve treatment response.
... Human PSCderived NPCs typically differentiate into neurons with a small proportion of glia. Current consensus suggests that this neuronal bias is due to the developmental immaturity of pluripotent derivatives and the fact that neurogenesis precedes gliogenesis during development (Miyata et al., 2010). Most of the experiments that have generated this consensus have thus far been carried out in atmospheric O 2 ; therefore, we assayed the effect of lowering the O 2 tension in human NPC cultures. ...
Hypoxia augments human embryonic stem cell (hESC) self-renewal via hypoxia-inducible factor 2?-activated OCT4 transcription. Hypoxia also increases the efficiency of reprogramming differentiated cells to a pluripotent-like state. Combined, these findings suggest that low O2 tension would impair the purposeful differentiation of pluripotent stem cells. Here, we show that low O2 tension and hypoxia-inducible factor (HIF) activity instead promote appropriate hESC differentiation. Through gain- and loss-of-function studies, we implicate O2 tension as a modifier of a key cell fate decision, namely whether neural progenitors differentiate toward neurons or glia. Furthermore, our data show that even transient changes in O2 concentration can affect cell fate through HIF by regulating the activity of MYC, a regulator of LIN28/let-7 that is critical for fate decisions in the neural lineage. We also identify key small molecules that can take advantage of this pathway to quickly and efficiently promote the development of mature cell types.
... While IPs do not share typical AP markers, such as Pax6, nestin and BLBP, they express the non-coding RNA Svet1 (Tarabykin et al. 2001) and the transcription factors Tbr2 (Englund et al. 2005), Cux 1 and Cux 2 (Nieto et al. 2004;Zimmer et al. 2004). It is worth noting that several transcription factors promote the specification of IPs (Miyata et al. 2010). APs that engage in the generation of IPs start to express AP2γ, a transcription factor required for Tbr2 expression by IPs. ...
The mammalian cerebral cortex is characterized by a complex histological organization that reflects the spatio-temporal stratifications of related stem and neural progenitor cells, which are responsible for the generation of distinct glial and neuronal subtypes during development. Some work has been done to shed light on the existing filiations between these progenitors as well as their respective contribution to cortical neurogenesis. The aim of the present review is to summarize the current views of progenitor hierarchy and relationship in the developing cortex and to further discuss future research directions that would help us to understand the molecular and cellular regulating mechanisms involved in cerebral corticogenesis.
In contrast to stage-specific transcription factors, the role of ubiquitous transcription factors in neuronal development remains a matter of scrutiny. Here, we demonstrated that a ubiquitous factor NF-Y is essential for neural progenitor maintenance during brain morphogenesis. Deletion of the NF-YA subunit in neural progenitors by using nestin-cre transgene in mice resulted in significant abnormalities in brain morphology, including a thinner cerebral cortex and loss of striatum during embryogenesis. Detailed analyses revealed a progressive decline in multiple neural progenitors in the cerebral cortex and ganglionic eminences, accompanied by induced apoptotic cell death and reduced cell proliferation. In neural progenitors, the NF-YA short isoform lacking exon 3 is dominant and co-expressed with cell cycle genes. ChIP-seq analysis from the cortex during early corticogenesis revealed preferential binding of NF-Y to the cell cycle genes, some of which were confirmed to be downregulated following NF-YA deletion. Notably, the NF-YA short isoform disappears and is replaced by its long isoform during neuronal differentiation. Forced expression of the NF-YA long isoform in neural progenitors resulted in a significant decline in neuronal count, possibly due to the suppression of cell proliferation. Collectively, we elucidated a critical role of the NF-YA short isoform in maintaining neural progenitors, possibly by regulating cell proliferation and apoptosis. Moreover, we identified an isoform switch in NF-YA within the neuronal lineage in vivo, which may explain the stage-specific role of NF-Y during neuronal development.
Folding of the cerebral cortex is a fundamental milestone of mammalian brain evolution associated with dramatic increases in size and complexity. Cortex folding takes place during embryonic and perinatal development and is important to optimize the functional organization and wiring of the brain, while allowing fitting a large cortex in a limited cranial volume. Cortex growth and folding are the result of complex cellular and mechanical processes that involve neural stem progenitor cells and their lineages, the migration and differentiation of neurons, and the genetic programs that regulate and fine-tune these processes. Here, we provide an updated overview of the most significant and recent advances in our understanding of developmental mechanisms regulating cortical gyrification.
The mammalian brain is probably the most fascinating and complex organ that has evolved over millions of years. In this chapter, we learn about some key genetic factors that control mammalian brain development with a focus on the cerebral cortex. Selected topics highlight oscillatory expression of neurogenic and proneural factors, the relationship between cell cycle control and cell fate and the spatiotemporal generation of neurons in the layered cortex. In a second part, we introduce the neural stem and progenitor cell types in the mammalian neocortex that potentially are the key recent inventions to distinguish higher evolved gyrencephalic from more primitive lissencephalic brains. We discuss the concepts and cellular mechanisms that might have led to neocortex expansion during evolution toward the primate brain.KeywordsNeural stem cellBrain developmentNeocortexRadial glial cellNeuroepithelial cellGyrencephalic
This first chapter provides an overview of the field of neurogenetics including:KeywordsNeurogeneticsNeurobiologyModel organisms
Neurodegenerative diseases comprise a wide range of age-related conditions, characterized by the loss of neurons leading to a progressive decline in brain function. Diverse cellular and animal models have enhanced our understanding of the molecular pathogenesis of different neurodegenerative diseases. However, failure to translate potential therapeutic findings from bench to bedside is primarily due to limitations of animal models and needs urgent attention. In this chapter, we discuss different neurodegenerative diseases and compare the different models commonly used in their study.KeywordsNeurodegenerative diseaseAlzheimer’s disease (AD)Parkinson’s disease (PD)Amyotrophic lateral sclerosis (ALS)Animal modelsInduced pluripotent stem cells (iPSCs)
Recent genetic studies of neurodevelopmental disorders point to synaptic proteins and ion channels as key contributors to disease pathogenesis. Although many of these proteins, such as the L-type calcium channel Cav1.2 or the postsynaptic scaffolding protein SHANK3, have well-studied functions in mature neurons, new evidence indicates that they may subserve novel, distinct roles in immature cells as the nervous system is assembled in prenatal development. Emerging tools and technologies, including single-cell sequencing and human cellular models of disease, are illuminating differential isoform utilization, spatiotemporal expression, and subcellular localization of ion channels and synaptic proteins in the developing brain compared with the adult, providing new insights into the regulation of developmental processes. We propose that it is essential to consider the temporally distinct and cell-specific roles of these proteins during development and maturity in our framework for understanding neuropsychiatric disorders.
During mammalian neocortical development, neural progenitor cells undergo sequential division to produce different types of progenies. Regulation of when and how many cells with a specific fate are produced from neural progenitor cells, i.e., 'temporal patterning' for cytogenesis, is crucial for the formation of the functional neocortex. Recently advanced techniques for transcriptome profiling at the single-cell level provide a solid basis to investigate the molecular nature underlying temporal patterning, including examining the necessity of cell-cycle progression. Evidence has indicated that cell-intrinsic programs and extrinsic cues coordinately regulate the timing of both the change in the division mode of neural progenitors from proliferative to neurogenic and their laminar fate transition from deep-layer to upper-layer neurons. Epigenetic modulation, transcriptional cascades, and post-transcriptional regulation are reported to function as cell-intrinsic programs, whereas extrinsic cues from the environment or surrounding cells supposedly function in a negative feedback or positive switching manner for temporal patterning. These findings suggest that neural progenitor cells have intrinsic temporal programs that can progress cell-autonomously and cell-cycle independently, while extrinsic cues play a critical role in tuning the temporal programs to let neural progenitor cells know the 'right' time to progress.
Neural progenitor cells hold significant promise in a variety of clinical settings. While both the brain and spinal cord harbor endogenous neural progenitor or stem cells, they typically are not capable of repopulating neural populations in case of injury or degenerative disease. In vitro systems for the culture of neural progenitors has come a long ways due to advances in the method development. Recently, many groups have shown that manipulation of the oxygen-sensing pathway leading to activation of hypoxia inducible factors (HIFs) that can influence the proliferation, differentiation or maturation of neural progenitors. Moreover, different oxygen concentrations appear to affect lineage specification of neural progenitors upon their differentiation in vitro. Here we summarize some of these studies in an attempt to direct effort towards implementation of best methods to advance the use of neural progenitors from basic development towards clinical application.
A tight regulation of neuron production is required to generate a functional cerebral cortex and is achieved by a proper balance between proliferation and differentiation of progenitor cells. Though the vitamin A (retinol) active derivative retinoic acid (RA) has been implicated as one of the signals acting during mammalian forebrain neurogenesis, its function at the onset of neurogenesis as well as during establishment of cortical layers and neuronal subtypes remains elusive. One limitation is that murine mutants for genes encoding key enzymes involved in RA synthesis die during early embryonic development. We analysed corticogenesis in Rdh10 null mutants, in which an RA deficiency is generated as the intracellular retinol to retinaldehyde conversion is abolished. When analysed at the latest stage before lethality occurs (embryonic day [E]13.5), the mutants show smaller telencephalic vesicles and the thickness of their cortical plate is strongly reduced. The first progenitors formed in the cortical plate are radial glial (RG) cells which generate neurons either directly, or through an indirect mechanism involving the production of intermediate neuronal progenitors (INPs) which then give rise to neurons. We show that in absence of RA, the RG progenitors proliferate less and prematurely produce neurons, leading to their depletion at E11.5. Furthermore, we could demonstrate that lack of RA impairs the generation of INPs at E13.5 and affects the cell cycle exit of progenitor cells during corticogenesis, altogether leading to a deficit in projection neurons and to microcephaly.
Down syndrome (DS) arises from triplication of genes on human chromosome 21 and is associated with anomalies in brain development such as reduced production of neurons and increased generation of astrocytes. Here, we show that differentiation of cortical progenitor cells into astrocytes is promoted by DYRK1A, a Ser/Thr kinase encoded on human chromosome 21. In the Ts1Cje mouse model of DS, increased dosage of DYRK1A augments the propensity of progenitors to differentiate into astrocytes. This tendency is associated with enhanced astrogliogenesis in the developing neocortex. We also find that overexpression of DYRK1A upregulates the activity of the astrogliogenic transcription factor STAT in wild-type progenitors. Ts1Cje progenitors exhibit elevated STAT activity, and depletion of DYRK1A in these cells reverses the deregulation of STAT. In sum, our findings indicate that potentiation of the DYRK1A-STAT pathway in progenitors contributes to aberrant astrogliogenesis in DS.
The temporal and spatial pattern of microglia colonization of the nervous system implies a role in neural cell proliferation and differentiation, as well as neurovascularization and postnatal myelination. Microglia established within the developing nervous system assume a neural-specific identity and contribute to key developmental events. Their association around blood vessels implicates them in development of the vascular system. A similar association has been reported for neural cell proliferation and associated phenotypic shifts, and also for cell fate differentiation to neuronal or glial phenotypes. These processes are accomplished by phagocytic activities, cell-cell contact relationships, and secretion of various factors. This chapter will provide an assessment of data currently available from in vivo and in vitro studies evaluating the dynamic, interactive nature of these processes throughout the progression of nervous system development. © 2014 Springer Science+Business Media New York. All rights reserved.
Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled as loss or an altered division orientation can promote overgrowth, alter tissue architecture and aberrant differentiation, and has been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.
Ethnopharmacological relevance:
Half a dozen of the currently accepted 135 Turnera species are used in traditional medicine, most notably Turnera diffusa Willd. ex Schult. which is one of the most highly appreciated plant aphrodisiacs. Other traditional uses of Turnera L. species include the treatment of anaemia, bronchitis, cough, diabetes, fever, fungal disease, gastrointestinal complaints, pain, pulmonary and respiratory diseases, skin disorders, and women׳s health problems. Additionally, Turnera species are used as abortives, expectorants, and laxatives.
Phytochemistry:
Flavonoids (22 different compounds), maltol glucoside, phenolics, cyanogenic glycosides (7 different compounds), monoterpenoids, sesquiterpenoids, triterpenoids, the polyterpene ficaprenol-11, fatty acids, and caffeine have been found in the genus Turnera.
Bioactivity:
Bioactivities experimentally proven for members of the genus Turnera encompass antianxiety, antiaromatase, antibacterial including antimycobacterial, antidiabetic, antioxidant, adapatogenic, antiobesity, antispasmodic, cytotoxic, gastroprotective, hepatoprotective, and aphrodisiac activities. Most of these activities have so far been investigated only in chemical, cell based, or animal assays. In contrast, the antiobesity activity was also investigated in a study on healthy human subjects and with a herbal preparation containing among other ingredients Turnera diffusa leaves. Moreover, the enhancement of female sexual function was assessed in humans; again the product contained besides Turnera diffusa other potentially bioactive ingredients. However, with only few exceptions, most of the traditional uses and the experimentally verified bioactivities can currently not be related to a particular compound or compound class. A notable exception is the flavonoid apigenin, which was identified animal experiments as the antinociceptive principle of Turnera diffusa.
Conclusion:
In this review, the current knowledge on ethnobotanical uses of members of the genus Turnera, the secondary metabolites reported from Turnera, and experimentally documented bioactivities from Turnera extracts and pure compounds derived from Turnera extracts are compiled. Moreover, some of the most interesting avenues for future research projects are being discussed briefly. These include in particular the aphrodisiac activity of Turnera diffusa and the antibiotic activity potentiating effect of Turnera ulmifolia L. against aminoglycoside resistant bacteria.
The 22q11.2 deletion syndrome (22q11DS) presents with medical and neuropsychiatric manifestations including neurocognitive deficits. Quantitative neurobehavioral measures linked to brain circuitry can help elucidate genetic mechanisms contributing to deficits. To establish the neurocognitive profile and neurocognitive 'growth charts', we compared cross-sectionally 137 individuals with 22q11DS ages 8-21 to 439 demographically matched non-deleted individuals with developmental delay (DD) and medical comorbidities and 443 typically developing (TD) participants. We administered a computerized neurocognitive battery that measures performance accuracy and speed in executive, episodic memory, complex cognition, social cognition and sensorimotor domains. The accuracy performance profile of 22q11DS showed greater impairment than DD, who were impaired relative to TD. Deficits in 22q11DS were most pronounced for face memory and social cognition, followed by complex cognition. Performance speed was similar for 22q11DS and DD, but 22q11DS individuals were differentially slower in face memory and emotion identification. The growth chart, comparing neurocognitive age based on performance relative to chronological age, indicated that 22q11DS participants lagged behind both groups from the earliest age assessed. The lag ranged from less than 1 year to over 3 years depending on chronological age and neurocognitive domain. The greatest developmental lag across the age range was for social cognition and complex cognition, with the smallest for episodic memory and sensorimotor speed, where lags were similar to DD. The results suggest that 22q11.2 microdeletion confers specific vulnerability that may underlie brain circuitry associated with deficits in several neuropsychiatric disorders, and therefore help identify potential targets and developmental epochs optimal for intervention.Molecular Psychiatry advance online publication, 21 January 2014; doi:10.1038/mp.2013.189.
In the central nervous system (CNS), the glial gap junctions are established among astrocytes (ASTs), oligodendrocytes (OLs), and/or between ASTs and OLs due to the expression of membrane proteins called connexins (Cxs). Together, the glial cells form a network of communicating cells that is important for the homeostasis of brain function for its involvement in the intercellular calcium wave propagation, exchange of metabolic substrates, cell proliferation, migration, and differentiation. Alternatively, Cxs are also involved in hemichannel function and thus participate in gliotransmission. In recent years, pathologic changes of oligodendroglia or demyelination found in transgenic mice with different subsets of Cxs or pharmacological insults suggest that glial Cxs may participate in the regulation of the myelination or remyelination processes. However, little is known about the underlying mechanisms. In this review, we will mainly focus on the functions of Cx-mediated gap junction channels, as well as hemichannels, in brain glial cells and discuss the way by which they impact myelination and remyelination. These aspects will be considered at the light of recent genetic and non-genetic studies related to demyelination and remyelination.
The fate of cortical progenitors, which progressively generate neurons and glial cells during development, is determined by temporally and spatially regulated signaling mechanisms. We found that the transcription factor Sip1 (Zfhx1b), which is produced at high levels in postmitotic neocortical neurons, regulates progenitor fate non-cell autonomously. Conditional deletion of Sip1 in young neurons induced premature production of upper-layer neurons at the expense of deep layers, precocious and increased generation of glial precursors, and enhanced postnatal astrocytogenesis. The premature upper-layer generation coincided with overexpression of the neurotrophin-3 (Ntf3) gene and upregulation of fibroblast growth factor 9 (Fgf9) gene expression preceded precocious gliogenesis. Exogenous application of Fgf9 to mouse cortical slices induced excessive generation of glial precursors in the germinal zone. Our data suggest that Sip1 restrains the production of signaling factors in postmitotic neurons that feed back to progenitors to regulate the timing of cell fate switch and the number of neurons and glial cells throughout corticogenesis.
Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. Here we show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex.
An important feature of the cerebral cortex is its layered organization, which is modulated in an area-specific manner. We found that the transcription factor AP2gamma regulates laminar fate in a region-specific manner. Deletion of AP2gamma (also known as Tcfap2c) during development resulted in a specific reduction of upper layer neurons in the occipital cortex, leading to impaired function and enhanced plasticity of the adult visual cortex. AP2gamma functions in apical progenitors, and its absence resulted in mis-specification of basal progenitors in the occipital cortex at the time at which upper layer neurons were generated. AP2gamma directly regulated the basal progenitor fate determinants Math3 (also known as Neurod4) and Tbr2, and its overexpression promoted the generation of layer II/III neurons in a time- and region-specific manner. Thus, AP2gamma acts as a regulator of basal progenitor fate, linking regional and laminar specification in the mouse developing cerebral cortex.
Expression of cyclins D1 (cD1) and D2 (cD2) in ventricular zone and subventricular zone (SVZ), respectively, suggests that a switch to cD2 could be a requisite step in the generation of cortical intermediate progenitor cells (IPCs). However, direct evidence is lacking. Here, cD1 or cD2 was seen to colabel subsets of Pax6-expressing radial glial cells (RGCs), whereas only cD2 colabeled with Tbr2. Loss of IPCs in cD2(-/-) embryonic cortex and analysis of expression patterns in mutant embryos lacking cD2 or Tbr2 indicate that cD2 is used as progenitors transition from RGCs to IPCs and is important for the expansion of the IPC pool. This was further supported by the laminar thinning, microcephaly, and selective reduction in the cortical SVZ population in the cD2(-/-)cortex. Cell cycle dynamics between embryonic day 14-16 in knock-out lines showed preserved parameters in cD1 mutants that induced cD2 expression, but absence of cD2 was not compensated by cD1. Loss of cD2 was associated with reduced proliferation and enhanced cell cycle exit in embryonic cortical progenitors, indicating a crucial role of cD2 for the support of cortical IPC divisions. In addition, knock-out of cD2, but not cD1, affected both G(1)-phase and also S-phase duration, implicating the importance of these phases for division cycles that expand the progenitor pool. That cD2 was the predominant D-cyclin expressed in the human SVZ at 19-20 weeks gestation indicated the evolutionary importance of cD2 in larger mammals for whom expansive intermediate progenitor divisions are thought to enable generation of larger, convoluted, cerebral cortices.
Each of the five cellular layers of the cerebral neocortex is composed of a specific number of a single predominant 'class' of projection neuron. The projection neuron class is defined by its unique morphology and axonal projections to other areas of the brain. Precursor cell populations lining the embryonic lateral ventricles produce the projection neurons. The mechanisms regulating precursor cell proliferation also regulate total numbers of neurons produced at specific developmental periods and destined to a specific neocortical layer. Because the newborn neurons migrate relatively long distances to reach their final layer destinations, it is often assumed that the mechanisms governing acquisition of neuronal-class-specific characteristics, many of which become evident after neuron production, are independent of the mechanisms governing neuron production. We review evidence that suggests that the two mechanisms might be linked via operations of Notch1 and p27(Kip1), molecules known to regulate precursor cell proliferation and neuron production.
Glial cells were long considered end products of neural differentiation, specialized supportive cells with an origin very different from that of neurons. New studies have shown that some glial cells--radial glia (RG) in development and specific subpopulations of astrocytes in adult mammals--function as primary progenitors or neural stem cells (NSCs). This is a fundamental departure from classical views separating neuronal and glial lineages early in development. Direct visualization of the behavior of NSCs and lineage-tracing studies reveal how neuronal lineages emerge. In development and in the adult brain, many neurons and glial cells are not the direct progeny of NSCs, but instead originate from transit amplifying, or intermediate, progenitor cells (IPCs). Within NSCs and IPCs, genetic programs unfold for generating the extraordinary diversity of cell types in the central nervous system. The timing in development and location of NSCs, a property tightly linked to their neuroepithelial origin, appear to be the key determinants of the types of neurons generated. Identification of NSCs and IPCs is critical to understand brain development and adult neurogenesis and to develop new strategies for brain repair.
Investigating the mechanisms controlling the asymmetric division of neocortical progenitors that generate neurones in the mammalian brain is crucial for understanding the abnormalities of cortical development. Partitioning of fate determinants is a key instructive step and components of the apical junctional complex (adherens junctions), including the polarity proteins PAR3 and aPKC as well as adhesion molecules such as N-cadherin, have been proposed to be candidate determinants. In this study, however, we found no correlation between the partitioning of N-cadherin and fate determination. Rather, we show that adherens junctions comprise three membrane domains, and that during asymmetrical division these are split such that both daughters retain the adhesive proteins that control cell position, but only one daughter inherits the polarity proteins along with the apical membrane. This provides a molecular explanation as to how both daughters remain anchored to the ventricular surface after mitosis, while adopting different fates.
In mammalian brain development, neuroepithelial cells act as progenitors that produce self-renewing and differentiating cells.
Recent technical advances in live imaging and gene manipulation now enable us to investigate how neural progenitors generate
the 2 different types of cells with unprecedented accuracy and resolution, shedding new light on the roles of epithelial structure
in cell fate decisions and also on the plasticity of neurogenesis.
The excitatory neurons of the mammalian cerebral cortex arise from asymmetric divisions of radial glial cells in the ventricular zone and symmetric division of intermediate progenitor cells (IPCs) in the subventricular zone (SVZ) of the embryonic cortex. Little is known about the microenvironment in which IPCs divide or whether a stem cell niche exists in the SVZ of the embryonic cortex. Recent evidence suggests that vasculature may provide a niche for adult stem cells but its role in development is less clear. We have investigated the vasculature in the embryonic cortex during neurogenesis and find that IPCs are spatially and temporally associated with blood vessels during cortical development. Intermediate progenitors mimic the pattern of capillaries suggesting patterns of angiogenesis and neurogenesis are coordinated during development. More importantly, we find that IPCs divide near blood vessel branch points suggesting that cerebral vasculature establishes a stem cell niche for intermediate progenitors in the SVZ. These data provide novel evidence for the presence of a neurogenic niche for intermediate progenitors in the embryonic SVZ and suggest blood vessels are important for proper patterning of neurogenesis.
The planar cell polarity (PCP) pathway organizes the cytoskeleton and polarizes cells within embryonic tissue. We investigate the relationship between PCP signaling and cell fate determination during asymmetric division of neural progenitors (NPs) in mouse embryos. The cortex of Lp/Lp (Loop-tail) mice deficient in the essential PCP mediator Vangl2, homologue of Drosophila melanogaster Strabismus (Stbm), revealed precocious differentiation of neural progenitors into early-born neurons at the expense of late-born neurons and glia. Although Lp/Lp NPs were easily maintained in vitro, they showed premature differentiation and loss of asymmetric distribution of Leu-Gly-Asn-enriched protein (LGN)/partner of inscuteable (Pins), a regulator of mitotic spindle orientation. Furthermore, we observed a decreased frequency in asymmetric distribution of the LGN target nuclear mitotic apparatus protein (NuMa) in Lp/Lp cortical progenitors in vivo. This was accompanied by an increase in the number of vertical cleavage planes typically associated with equal daughter cell identities. These findings suggest that Stbm/Vangl2 functions to maintain cortical progenitors and regulates mitotic spindle orientation during asymmetric divisions in the vertebrate brain.
In the developing CNS, subtypes of neurons and glial cells are generated according to a schedule that is defined by cell-intrinsic mechanisms that function at the progenitor-cell level. However, no critical molecular switch for the temporal specification of CNS progenitor cells has been identified. We found that chicken ovalbumin upstream promoter-transcription factor I and II (Coup-tfI and Coup-tfII, also known as Nr2f1 and Nr2f2) are required for the temporal specification of neural stem/progenitor cells (NSPCs), including their acquisition of gliogenic competence, as demonstrated by their responsiveness to gliogenic cytokines. COUP-TFI and II are transiently co-expressed in the ventricular zone of the early embryonic CNS. The double knockdown of Coup-tfI/II in embryonic stem cell (ESC)-derived NSPCs and the developing mouse forebrain caused sustained neurogenesis and the prolonged generation of early-born neurons. These findings reveal a part of the timer mechanisms for generating diverse types of neurons and glial cells during CNS development.
T-brain gene-2 (Tbr2) is specifically expressed in the intermediate (basal) progenitor cells (IPCs) of the developing cerebral cortex; however, its function in this biological context has so far been overlooked due to the early lethality of Tbr2 mutant embryos. Conditional ablation of Tbr2 in the developing forebrain resulted in the loss of IPCs and their differentiated progeny in mutant cortex. Intriguingly, early loss of IPCs led to a decrease in cortical surface expansion and thickness with a neuronal reduction observed in all cortical layers. These findings suggest that IPC progeny contribute to the correct morphogenesis of each cortical layer. Our observations were confirmed by tracing Tbr2+ IPC cell fate using Tbr2::GFP transgenic mice. Finally, we demonstrated that misexpression of Tbr2 is sufficient to induce IPC identity in ventricular radial glial cells (RGCs). Together, these findings identify Tbr2 as a critical factor for the specification of IPCs during corticogenesis.
The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Here we demonstrate that conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ.
Epigenetic changes are thought to lead to alterations in the property of cells, such as differentiation potential. Neural precursor cells (NPCs) differentiate only into neurons in the midgestational brain, yet they become able to generate astrocytes in the late stage of development. This differentiation-potential switch could be explained by epigenetic changes, since the promoters of astrocyte-specific marker genes, glial fibrillary acidic protein (Gfap) and S100beta, have been shown to become demethylated in late-stage NPCs prior to the onset of astrocyte differentiation; however, whether demethylation occurs generally in other astrocyctic genes remains unknown. Here we analyzed DNA methylation changes in mouse NPCs between the mid-(E11.5) and late (E14.5) stage of development by a genome-wide DNA methylation profiling method using microarrays and found that many astrocytic genes are demethylated in late-stage NPCs, enabling the cell to become competent to express these genes. Although these genes are already demethylated in late-stage NPCs, they are not expressed until cells differentiate into astrocytes. Thus, late-stage NPCs have epigenetic potential which can be realized in their expression after astrocyte differentiation.
Cellular diversity of the brain is largely attributed to the spatial and temporal heterogeneity of progenitor cells. In mammalian cerebral development, it has been difficult to determine how heterogeneous the neural progenitor cells are, owing to dynamic changes in their nuclear position and gene expression. To address this issue, we systematically analyzed the cDNA profiles of a large number of single progenitor cells at the mid-embryonic stage in mouse. By cluster analysis and in situ hybridization, we have identified a set of genes that distinguishes between the apical and basal progenitors. Despite their relatively homogeneous global gene expression profiles, the apical progenitors exhibit highly variable expression patterns of Notch signaling components, raising the possibility that this causes the heterogeneous division patterns of these cells. Furthermore, we successfully captured the nascent state of basal progenitor cells. These cells are generated shortly after birth from the division of the apical progenitors, and show strong expression of the major Notch ligand delta-like 1, which soon fades away as the cells migrate in the ventricular zone. We also demonstrated that attenuation of Notch signals immediately induces differentiation of apical progenitors into nascent basal progenitors. Thus, a Notch-dependent feedback loop is likely to be in operation to maintain both progenitor populations.
In vertebrates, Notch signaling is generally thought to inhibit neural differentiation. However, whether Notch can also promote specific early cell fates in this context is unknown. We introduced activated Notch1 (NIC) into the mouse forebrain, before the onset of neurogenesis, using a retroviral vector and ultrasound imaging. During embryogenesis, NIC-infected cells became radial glia, the first specialized cell type evident in the forebrain. Thus, rather than simply inhibiting differentiation, Notch1 signaling promoted the acquisition of an early cellular phenotype. Postnatally, many NIC-infected cells became periventricular astrocytes, cells previously shown to be neural stem cells in the adult. These results suggest that Notch1 promotes radial glial identity during embryogenesis, and that radial glia may be lineally related to stem cells in the adult nervous system.
Radial glial cells and astrocytes function to support the construction and maintenance, respectively, of the cerebral cortex. However, the mechanisms that determine how radial glial cells are established, maintained, and transformed into astrocytes in the cerebral cortex are not well understood. Here, we show that neuregulin-1 (NRG-1) exerts a critical role in the establishment of radial glial cells. Radial glial cell generation is significantly impaired in NRG mutants, and this defect can be rescued by exogenous NRG-1. Down-regulation of expression and activity of erbB2, a member of the NRG-1 receptor complex, leads to the transformation of radial glial cells into astrocytes. Reintroduction of erbB2 transforms astrocytes into radial glia. The activated form of the Notch1 receptor, which promotes the radial glial phenotype, activates the erbB2 promoter in radial glial cells. These results suggest that developmental changes in NRG-1-erbB2 interactions modulate the establishment of radial glia and contribute to their appropriate transformation into astrocytes.
Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of beta-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the beta-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized beta-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development.
Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.
The Notch and fibroblast growth factor (FGF) pathways both regulate cell fate specification during mammalian neural development. We have shown previously that Notch1 activation in the murine forebrain promotes radial glial identity. This result, together with recent evidence that radial glia can be progenitors, suggested that Notch1 signaling might promote progenitor and radial glial character simultaneously. Consistent with this idea, we found that in addition to promoting radial glial character in vivo , activated Notch1 (ActN1) increased the frequency of embryonic day 14.5 (E14.5) ganglionic eminence (GE) progenitors that grew into neurospheres in FGF2. Constitutive activation of C-promoter binding factor (CBF1), a Notch pathway effector, also increased neurosphere frequency in FGF2, suggesting that the effect of Notch1 on FGF responsiveness is mediated by CBF1.
The observation that ActN1 promoted FGF responsiveness in telencephalic progenitors prompted us to examine the effect of FGF pathway activation in vivo . We focused on FGFR2 because it is expressed in radial glia in the GEs where ActN1 increases FGF2 neurosphere frequency, but not in the septum where it does not. Like ActN1, activated FGFR2 (ActFGFR2) promoted radial glial character in vivo . However, unlike ActN1, ActFGFR2 did not enhance neurosphere frequency at E14.5. Additional analysis demonstrated that, unexpectedly, neither ActFGFR2 nor ActFGFR1 could replace the need for ligand in promoting neurosphere proliferation. This study suggests that telencephalic progenitors with radial glial morphology are maintained by interactions between the Notch and FGF pathways, and that the mechanisms by which FGF signaling promotes radial glial character in vivo and progenitor proliferation in vitro can be uncoupled.
Radial glia function during CNS development both as neural progenitors and as a scaffolding supporting neuronal migration. To elucidate pathways involved in these functions, we mapped in vivo the promoter for Blbp, a radial glial gene. We show here that a binding site for the Notch effector CBF1 is essential for all Blbp transcription in radial glia, and that BLBP expression is significantly reduced in the forebrains of mice lacking the Notch1 and Notch3 receptors. These results identify Blbp as the first predominantly CNS-specific Notch target gene and suggest that it mediates some aspects of Notch signaling in radial glia.
DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. We report here that conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1-/- NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. Our study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation.
The size of brain regions depends on the balance between proliferation and differentiation. During development of the mouse cerebral cortex, ventricular zone (VZ) progenitors, neuroepithelial and radial glial cells, enlarge the progenitor pool by proliferative divisions, while basal progenitors located in the subventricular zone (SVZ) mostly divide in a differentiative mode generating two neurons. These differences correlate to the existence of an apico-basal polarity in VZ, but not SVZ, progenitors. Only VZ progenitors possess an apical membrane domain at which proteins of the Par complex are strongly enriched. We describe a prominent decrease in the amount of Par-complex proteins at the apical surface during cortical development and examine the role of these proteins by gain- and loss-of-function experiments. Par3 (Pard3) loss-of-function led to premature cell cycle exit, reflected in reduced clone size in vitro and the restriction of the progeny to the lower cortical layers in vivo. By contrast, Par3 or Par6 (Pard6alpha) overexpression promoted the generation of Pax6+ self-renewing progenitors in vitro and in vivo and increased the clonal progeny of single progenitors in vitro. Time-lapse video microscopy revealed that a change in the mode of cell division, rather than an alteration of the cell cycle length, causes the Par-complex-mediated increase in progenitors. Taken together, our data demonstrate a key role for the apically located Par-complex proteins in promoting self-renewing progenitor cell divisions at the expense of neurogenic differentiation in the developing cerebral cortex.
During mammalian development, neuroepithelial cells function as mitotic progenitors, which self-renew and generate neurons. Although spindle orientation is important for such polarized cells to undergo symmetric or asymmetric divisions, its role in mammalian neurogenesis remains unclear. Here we show that control of spindle orientation is essential in maintaining the population of neuroepithelial cells, but dispensable for the decision to either proliferate or differentiate. Knocking out LGN, (the G protein regulator), randomized the orientation of normally planar neuroepithelial divisions. The resultant loss of the apical membrane from daughter cells frequently converted them into abnormally localized progenitors without affecting neuronal production rate. Furthermore, overexpression of Inscuteable to induce vertical neuroepithelial divisions shifted the fate of daughter cells. Our results suggest that planar mitosis ensures the self-renewal of neuroepithelial progenitors by one daughter inheriting both apical and basal compartments during neurogenesis.
Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.
During neocortical development, neural precursor cells (NPCs, or neural stem cells) produce neurons first and astrocytes later. Although the timing of the fate switch from neurogenic to astrogenic is critical for determining the number of neurons, the mechanisms are not fully understood. Here, we show that the polycomb group complex (PcG) restricts neurogenic competence of NPCs and promotes the transition of NPC fate from neurogenic to astrogenic. Inactivation of PcG by knockout of the Ring1B or Ezh2 gene or Eed knockdown prolonged the neurogenic phase of NPCs and delayed the onset of the astrogenic phase. Moreover, PcG was found to repress the promoter of the proneural gene neurogenin1 in a developmental-stage-dependent manner. These results demonstrate a role of PcG: the temporal regulation of NPC fate.
During mouse embryonic development, neural progenitors lengthen the G1 phase of the cell cycle and this has been suggested to be a cause, rather than a consequence, of neurogenesis. To investigate whether G1 lengthening alone may cause the switch of cortical progenitors from proliferation to neurogenesis, we manipulated the expression of cdk/cyclin complexes and found that cdk4/cyclinD1 overexpression prevents G1 lengthening without affecting cell growth, cleavage plane, or cell cycle synchrony with interkinetic nuclear migration. Specifically, overexpression of cdk4/cyclinD1 inhibited neurogenesis while increasing the generation and expansion of basal (intermediate) progenitors, resulting in a thicker subventricular zone and larger surface area of the postnatal cortex originating from cdk4/cyclinD1-transfected progenitors. Conversely, lengthening of G1 by cdk4/cyclinD1-RNAi displayed the opposite effects. Thus, G1 lengthening is necessary and sufficient to switch neural progenitors to neurogenesis, and overexpression of cdk4/cyclinD1 can be used to increase progenitor expansion and, perhaps, cortical surface area.
Asymmetric cell division of radial glial progenitors produces neurons while allowing self-renewal; however, little is known about the mechanism that generates asymmetry in daughter cell fate specification. Here, we found that mammalian partition defective protein 3 (mPar3), a key cell polarity determinant, exhibits dynamic distribution in radial glial progenitors. While it is enriched at the lateral membrane domain in the ventricular endfeet during interphase, mPar3 becomes dispersed and shows asymmetric localization as cell cycle progresses. Either removal or ectopic expression of mPar3 prevents radial glial progenitors from dividing asymmetrically yet generates different outcomes in daughter cell fate specification. Furthermore, the expression level of mPar3 affects Notch signaling, and manipulations of Notch signaling or Numb expression suppress mPar3 regulation of radial glial cell division and daughter cell fate specification. These results reveal a critical molecular pathway underlying asymmetric cell division of radial glial progenitors in the mammalian neocortex.
Radial glia (RG), the progenitors of cortical neurons and basal progenitors (BPs), differentiate from neuroepithelial cells (NCs) with stem cell properties. We show that the morphogen Fgf10 is transiently expressed by NCs coincident with the transition period of NC differentiation into RG. Targeted deletion of Fgf10 delays RG differentiation, whereas overexpression has opposing effects. Delayed RG differentiation in Fgf10 mutants occurs selectively in rostral cortex, paralleled by an extended period of symmetric NC divisions increasing progenitor number, coupled with delayed and initially diminished production of neurons and BPs. RG eventually differentiate in excess number and overproduce neurons and BPs rostrally resulting in tangential expansion of frontal areas and increased laminar thickness. Thus, transient Fgf10 expression regulates timely differentiation of RG, and through this function, determines both length of the early progenitor expansion phase and onset of neurogenesis and ultimately the number of progenitors and neurons fated to specific cortical areas.
The mechanisms that regulate symmetric, proliferative divisions versus asymmetric, neurogenic divisions of mammalian neural precursors are still not well understood. We found that Lfc (Arhgef2), a Rho-specific guanine nucleotide exchange factor that interacts with spindle microtubules, and its negative regulator Tctex-1 (Dynlt1) determine the genesis of neurons from precursors in the embryonic murine cortex. Specifically, genetic knockdown of Arhgef2 in cortical precursors either in culture or in vivo inhibited neurogenesis and maintained cells as cycling radial precursors. Conversely, genetic knockdown of Dynlt1 in radial precursors promoted neurogenesis and depleted cycling cortical precursors. Coincident silencing of these two genes indicated that Tctex-1 normally inhibits the genesis of neurons from radial precursors by antagonizing the proneurogenic actions of Lfc. Moreover, Lfc and Tctex-1 were required to determine the orientation of mitotic precursor cell divisions in vivo. Thus, Lfc and Tctex-1 interact to regulate cortical neurogenesis, potentially by regulating mitotic spindle orientation.
Spatial and temporal specification of neural progenitor cells is integral to their production of a wide variety of central nervous system (CNS) cells. For a given region, cells arise on a precise and predictable temporal schedule, with sub-types of neurons appearing in a defined order, followed by glial cell generation. Single cell studies have shown that the timing of cell generation can be encoded within individual early progenitor cells as a cell-intrinsic program. Environmental cues are important modulators of this program, allowing it to unfold and coordinating the process within the embryo. Here we review recent findings on the molecular mechanisms of epigenetic and transcription factor regulation, which are involved in temporal specification of CNS stem cells.
In the mouse neocortex, neural progenitor cells generate both differentiating neurons and daughter cells that maintain progenitor fate. Here, we show that the TRIM-NHL protein TRIM32 regulates protein degradation and microRNA activity to control the balance between those two daughter cell types. In both horizontally and vertically dividing progenitors, TRIM32 becomes polarized in mitosis and is concentrated in one of the two daughter cells. TRIM32 overexpression induces neuronal differentiation while inhibition of TRIM32 causes both daughter cells to retain progenitor cell fate. TRIM32 ubiquitinates and degrades the transcription factor c-Myc but also binds Argonaute-1 and thereby increases the activity of specific microRNAs. We show that Let-7 is one of the TRIM32 targets and is required and sufficient for neuronal differentiation. TRIM32 is the mouse ortholog of Drosophila Brat and Mei-P26 and might be part of a protein family that regulates the balance between differentiation and proliferation in stem cell lineages.
During midgestation, mammalian neural precursor cells (NPCs) differentiate only into neurons. Generation of astrocytes is prevented at this stage, because astrocyte-specific gene promoters are methylated. How the subsequent switch from suppression to expression of astrocytic genes occurs is unknown. We show in this study that Notch ligands are expressed on committed neuronal precursors and young neurons in mid-gestational telencephalon, and that neighboring Notch-activated NPCs acquire the potential to become astrocytes. Activation of the Notch signaling pathway in midgestational NPCs induces expression of the transcription factor nuclear factor I, which binds to astrocytic gene promoters, resulting in demethylation of astrocyte-specific genes. These findings provide a mechanistic explanation for why neurons come first: committed neuronal precursors and young neurons potentiate remaining NPCs to differentiate into the next cell lineage, astrocytes.
To understand the cellular and molecular mechanisms regulating cytogenesis within the neocortical ventricular zone, we examined at high resolution the spatiotemporal expression patterns of Ngn2 and Tbr2. Individually DiI-labeled daughter cells were tracked from their birth in slice cultures and immunostained for Ngn2 and Tbr2. Both proteins were initially absent from daughter cells during the first 2 h. Ngn2 expression was then initiated asymmetrically in one of the daughter cells, with a bias towards the apical cell, followed by a similar pattern of expression for Tbr2, which we found to be a direct target of Ngn2. How this asymmetric Ngn2 expression is achieved is unclear, but gamma-secretase inhibition at the birth of daughter cells resulted in premature Ngn2 expression, suggesting that Notch signaling in nascent daughter cells suppresses a Ngn2-Tbr2 cascade. Many of the nascent cells exhibited pin-like morphology with a short ventricular process, suggesting periventricular presentation of Notch ligands to these cells.
During the neurogenic phase of mammalian brain development, only a subpopulation of neural precursor cells (NPCs) differentiates into neurons. The mechanisms underlying this selection remain unclear. Here we provide evidence that the Notch-Delta pathway plays an important role in this selection in the developing mouse telencephalon. We found that the expression patterns of the Notch ligand delta-like 1 (Dll1) and of the active form of Notch1 were mutually exclusive and segregated into distinct NPC subpopulations in the ventricular zone of the telencephalon. When Dll1 was overexpressed in a small, but not a large, proportion of NPCs, these cells underwent neuronal differentiation in vitro and in vivo. This Dll1-induced neuronal differentiation did not occur when cells were plated at lower densities in an in vitro culture. Importantly, conditional deletion of the Dll1 gene in a small proportion of NPCs reduced neurogenesis in vivo, whereas deletion in a large proportion promoted premature neurogenesis. These results support the notion that different levels of Dll1 expression determine the fate of NPCs through cell-cell interactions, most likely through the Notch-Delta lateral inhibitory signaling pathway, thus contributing to the selection of differentiating cells.
Basal (intermediate) progenitors are the major source of neurons in the mammalian neocortex. The molecular machinery governing basal progenitor biogenesis is unknown. Here, we show that the zinc-finger transcription factor Insm1 (insulinoma-associated 1) is expressed specifically in progenitors undergoing neurogenic divisions, has a panneurogenic role throughout the brain, and promotes basal progenitor formation in the neocortex. Mouse embryos lacking Insm1 contained half the number of basal progenitors and showed a marked reduction in cortical plate radial thickness. Forced premature expression of Insm1 in neuroepithelial cells resulted in their mitosis occurring at the basal (rather than apical) side of the ventricular zone and induced expression of the basal progenitor marker Tbr2. Remarkably, these cells remained negative for Tis21, a marker of neurogenic progenitors, and did not generate neurons but underwent self-amplification. Our data imply that Insm1 is involved in the generation and expansion of basal progenitors, a hallmark of neocortex evolution.
The switch of neural stem and progenitor cells from proliferation to differentiation during development is a crucial determinant of brain size. This switch is intimately linked to the architecture of the two principal classes of neural stem and progenitor cells, the apical (neuroepithelial, radial glial) and basal (intermediate) progenitors, which in turn is crucial for their symmetric versus asymmetric divisions. Focusing on the developing rodent neocortex, we discuss here recent advances in understanding the cell biology of apical and basal progenitors, place key regulatory molecules into subcellular context, and highlight their roles in the control of proliferation versus differentiation.
The more than 1000-fold increase in the cortical surface without a comparable increase in its thickness during mammalian evolution is explained in the context of the radial-unit hypothesis of cortical development. According to the proposed model, cortical expansion is the result of changes in proliferation kinetics that increase the number of radial columnar units without changing the number of neurons within each unit significantly. Thus, mutation of a regulatory gene(s) that controls the timing and ratio of symmetric and asymmetric modes of cell divisions in the proliferative zone, coupled with radial constraints in the distribution of migrating neurons, could create an expanded cortical plate with enhanced capacity for establishing new patterns of connectivity that are validated through natural selection.
The author thanks J. Joseph, G. Holmes, M. Berg, and E. Miyawaki for providing figures; T. Chae, W. B. Dobyns, J. Gleeson, G. Mochida, and E. Monuki for thoughtful comments on the manuscript; and W. B. D. and members of the Walsh lab for many stimulating discussions about human malformations. Assistance with the preparation of the figures came from Katie Lee. Research in the author’s lab is supported by grants from the Human Frontier Science Program, the NINDS (RO1 NS35129, RO1 NS32457, RO1 NS38097, and PO1 NS38289), the National Alliance for Autism Research, the National Alliance for Research in Schizophrenia and Depression, and the Mental Retardation Research Center at Children’s Hospital, Boston.
The mechanisms by which neural stem cells give rise to neurons, astrocytes, or oligodendrocytes are beginning to be elucidated. However, it is not known how the specification of one cell lineage results in the suppression of alternative fates. We find that in addition to inducing neurogenesis, the bHLH transcription factor neurogenin (Ngn1) inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis. Thus, two distinct mechanisms are involved in the activation and suppression of gene expression during cell-fate specification by neurogenin.
Astrocyte differentiation, which occurs late in brain development, is largely dependent on the activation of a transcription factor, STAT3. We show that astrocytes, as judged by glial fibrillary acidic protein (GFAP) expression, never emerge from neuroepithelial cells on embryonic day (E) 11.5 even when STAT3 is activated, in contrast to E14.5 neuroepithelial cells. A CpG dinucleotide within a STAT3 binding element in the GFAP promoter is highly methylated in E11.5 neuroepithelial cells, but is demethylated in cells responsive to the STAT3 activation signal to express GFAP. This CpG methylation leads to inaccessibility of STAT3 to the binding element. We suggest that methylation of a cell type-specific gene promoter is a pivotal event in regulating lineage specification in the developing brain.
The functions of Wingless-Int (Wnt) signaling, studied intensely in embryonic brain development, have been comparatively little investigated in the postnatal brain. We report remarkably patterned gene expression of Wnt signaling components in postnatal mouse cerebral cortex, lasting into young adulthood. Wnt genes are expressed in gene-specific regional and lamina patterns in each of the major subdivisions of the cerebral cortex: the olfactory bulb (OB), the hippocampal formation, and the neocortex. Genes encoding Frizzled (Fz) Wnt receptors, or secreted Frizzled-related proteins (sFrps), are also expressed in regional and lamina patterns. These findings suggest that Wnt signaling is active and regulated in the postnatal cortex and that different cortical cell populations have varying requirements for a Wnt signal. The OB, in particular, shows gene expression of a large variety of Wnt signaling components, making it a prime target for future functional studies. The penultimate components of the canonical Wnt pathway are the Tcf/Lef1 transcription factors, which regulate transcription of Wnt signaling target genes. Surprisingly, we found little Tcf/Lef1 expression in the postnatal neocortex. These observations suggest that noncanonical Wnt pathways predominate, which will require functional testing. However, Lef1 is widely expressed in the dorsal thalamus, and Wnt ligands and receptors are expressed, respectively, in cortical areas and thalamic nuclei that are interconnected. Thus, canonical Wnt signaling could be utilized in a major cortical input by Fz- and Lef1-expressing thalamic cells that innervate the Wnt-expressing cortex.
Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete.
During the development of the mammalian central nervous system, neural stem cells and their derivative progenitor cells generate neurons by asymmetric and symmetric divisions. The proliferation versus differentiation of these cells and the type of division are closely linked to their epithelial characteristics, notably, their apical-basal polarity and cell-cycle length. Here, we discuss how these features change during development from neuroepithelial to radial glial cells, and how this transition affects cell fate and neurogenesis.
Embryonic multipotent neural precursors are exposed to extracellular signals instructing them to adopt different fates, neuronal or glial. However, the mechanisms by which precursors integrate these signals to make timely fate choices remained undefined. Here we show that direct nuclear signaling by a receptor tyrosine kinase inhibits the responses of precursors to astrocyte differentiation factors while maintaining their neurogenic potential. Upon neuregulin-induced activation and presenilin-dependent cleavage of ErbB4, the receptor's intracellular domain forms a complex with TAB2 and the corepressor N-CoR. This complex undergoes nuclear translocation and binds promoters of astrocytic genes, repressing their expression. Consistent with this observation, astrogenesis occurs precociously in ErbB4 knockout mice. Our studies define how presenilin-dependent nuclear signaling by a receptor tyrosine kinase directly regulates gene transcription and cell fate. This pathway could be of importance for neural stem cell biology and for understanding the pathogenesis of Alzheimer's disease.
During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neural circuitry, and subsequent matching of glial numbers and position to that circuitry. Here, we have reviewed work addressing the mechanisms underlying this timed cell genesis, with a particular focus on the developing cortex. These studies have defined an intriguing interplay between intrinsic epigenetic status, transcription factors, and environmental cues, all of which work together to establish this fascinating and complex biological timing mechanism.
A fundamental feature of neural development in vertebrates is that different cell types are generated in a precise temporal sequence, first neurons, followed by oligodendrocytes and astrocytes. The mechanisms underlying these remarkable changes in progenitor fate during development are not well understood, but are thought to include both changes in the intrinsic properties of neural progenitors and changes in their signaling environment. I discuss the mechanisms that control the specification of neuronal, astroglial and oligodendroglial fates, focusing on the mammalian telencephalon, one of the most extensively used models to study neural specification mechanisms in vertebrates. I first consider the multiple extracellular signals that have been implicated in neural fate specification. Their roles are often complex, with the same signals having different effects at different developmental stages, and different signaling pathways interacting extensively. The selection of a particular cell fate ultimately results from the integration of multiple signals. Signaling pathways regulate cell fates by modulating the expression and activity of numerous transcription factors in neural stem cells. I discuss how transcription factors also act in a combinatorial manner to determine progenitor fates, with individual factors promoting the generation of one or two cell types and repressing alternative fate(s). Finally, I discuss the many levels of regulation involved in preventing premature astrocyte differentiation during neurogenesis, and later on in controlling the transition from neurogenesis to gliogenesis.
Intermediate progenitor cells (IPCs) are a type of neurogenic transient amplifying cells in the developing cerebral cortex. IPCs divide symmetrically at basal (abventricular) positions in the neuroepithelium to produce pairs of new neurons or, in amplifying divisions, pairs of new IPCs. In contrast, radial unit progenitors (neuroepithelial cells and radial glia) divide at the apical (ventricular) surface and produce only single neurons or single IPCs by asymmetric division, or self-amplify by symmetric division. Histologically, IPCs are most prominent during the middle and late stages of neurogenesis, when they accumulate in the subventricular zone, a progenitor compartment linked to the genesis of upper neocortical layers (II-IV). Nevertheless, IPCs are present throughout cortical neurogenesis and produce neurons for all layers. In mice, changes in the abundance of IPCs caused by mutations of Pax6, Ngn2, Id4 and other genes are associated with parallel changes in cortical thickness but not surface area. In gyrencephalic brains, IPCs may play broader roles in determining not only laminar thickness, but also cortical surface area and gyral patterns. We propose that regulation of IPC genesis and amplification across developmental stages and regional subdivisions modulates laminar neurogenesis and contributes to the cytoarchitectonic differentiation of cortical areas.
Neocortical precursor cells undergo symmetric and asymmetric divisions while producing large numbers of diverse cortical cell types. In Drosophila, cleavage plane orientation dictates the inheritance of fate-determinants and the symmetry of newborn daughter cells during neuroblast cell divisions. One model for predicting daughter cell fate in the mammalian neocortex is also based on cleavage plane orientation. Precursor cell divisions with a cleavage plane orientation that is perpendicular with respect to the ventricular surface (vertical) are predicted to be symmetric, while divisions with a cleavage plane orientation that is parallel to the surface (horizontal) are predicted to be asymmetric neurogenic divisions. However, analysis of cleavage plane orientation at the ventricle suggests that the number of predicted neurogenic divisions might be insufficient to produce large amounts of cortical neurons. To understand factors that correlate with the symmetry of cell divisions, we examined rat neocortical precursor cells in situ through real-time imaging, marker analysis, and electrophysiological recordings. We find that cleavage plane orientation is more closely associated with precursor cell type than with daughter cell fate, as commonly thought. Radial glia cells in the VZ primarily divide with a vertical orientation throughout cortical development and undergo symmetric or asymmetric self-renewing divisions depending on the stage of development. In contrast, most intermediate progenitor cells divide in the subventricular zone with a horizontal orientation and produce symmetric daughter cells. We propose a model for predicting daughter cell fate that considers precursor cell type, stage of development, and the planar segregation of fate determinants.
Expression of the Notch effector gene Hes1 is required for maintenance of neural progenitors in the embryonic brain, but persistent and high levels of Hes1 expression inhibit proliferation and differentiation of these cells. Here, by using a real-time imaging method, we found that Hes1 expression dynamically oscillates in neural progenitors. Furthermore, sustained overexpression of Hes1 downregulates expression of proneural genes, Notch ligands, and cell cycle regulators, suggesting that their proper expression depends on Hes1 oscillation. Surprisingly, the proneural gene Neurogenin2 (Ngn2) and the Notch ligand Delta-like1 (Dll1) are also expressed in an oscillatory manner by neural progenitors, and inhibition of Notch signaling, a condition known to induce neuronal differentiation, leads to downregulation of Hes1 and sustained upregulation of Ngn2 and Dll1. These results suggest that Hes1 oscillation regulates Ngn2 and Dll1 oscillations, which in turn lead to maintenance of neural progenitors by mutual activation of Notch signaling.