Beta-Catenin Signaling Negatively Regulates Intermediate Progenitor Population Numbers in the Developing Cortex

Intermediate progenitor cells constitute a second proliferative cell type in the developing mammalian cerebral cortex. Little is known about the factors that govern the production of intermediate progenitors. Although persistent expression of stabilized β-catenin was found to delay the maturation of radial glial progenitors into intermediate progenitors, the relationship between β-catenin signaling and intermediate progenitors remains poorly understood. Using a transgenic reporter mouse for Axin2, a direct target of Wnt/β-catenin signaling, we observed that β-catenin signaling is decreased in intermediate progenitor cells relative to radial glial progenitors. Conditional deletion of β-catenin from mouse cortical neural progenitors increased intermediate progenitor numbers, while conditional expression of stabilized β-catenin reduced the intermediate progenitor population. Together, these findings provide evidence that β-catenin signaling in radial progenitors negatively regulates intermediate progenitor cell number during cortical development.     

Progressive restriction in fate potential by neural progenitors during cerebral cortical development

During early stages of cerebral cortical development, progenitor cells in the ventricular zone are multipotent, producing neurons of many layers over successive cell divisions. The laminar fate of their progeny depends on environmental cues to which the cells respond prior to mitosis. By the end of neurogenesis, however, progenitors are lineally committed to producing upper-layer neurons. Here we assess the laminar fate potential of progenitors at a middle stage of cortical development. The progenitors of layer 4 neurons were first transplanted into older brains in which layer 2/3 was being generated. The transplanted neurons adopted a laminar fate appropriate for the new environment (layer 2/3), revealing that layer 4 progenitors are multipotent. Mid-stage progenitors were then transplanted into a younger environment, in which layer 6 neurons were being generated. The transplanted neurons bypassed layer 6, revealing that layer 4 progenitors have a restricted fate potential and are incompetent to respond to environmental cues that trigger layer 6 production. Instead, the transplanted cells migrated to layer 4, the position typical of their origin, and also to layer 5, a position appropriate for neither the host nor the donor environment. Because layer 5 neurogenesis is complete by the stage that progenitors were removed for transplantation, restrictions in laminar fate potential must lag behind the final production of a cortical layer. These results suggest that a combination of intrinsic and environmental cues controls the competence of cortical progenitor cells to produce neurons of different layers.     

Live imaging at the onset of cortical neurogenesis reveals differential appearance of the neuronal phenotype in apical versus basal progenitor progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin-driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors.     

Reelin sets the pace of neocortical neurogenesis

Migration of neurons during cortical development is often assumed to rely on purely post-proliferative reelin signaling. However, Notch signaling, long known to regulate neural precursor formation and maintenance, is required for the effects of reelin on neuronal migration. Here, we show that reelin gain-of-function causes a higher expression of Notch target genes in radial glia and accelerates the production of both neurons and intermediate progenitor cells. Converse alterations correlate with reelin loss-of-function, consistent with reelin controlling Notch signaling during neurogenesis. Ectopic expression of reelin in isolated clones of progenitors causes a severe reduction in neuronal differentiation. In mosaic cell cultures, reelin-primed progenitor cells respond to wild-type cells by further decreasing neuronal differentiation, consistent with an increased sensitivity to lateral inhibition. These results indicate that reelin and Notch signaling cooperate to set the pace of neocortical neurogenesis, a prerequisite for proper neuronal migration and cortical layering.     

Neocortex expansion in development and evolution—from genes to progenitor cell biology

The evolutionary expansion of the neocortex, the seat of higher cognitive functions in humans, is primarily due to an increased and prolonged proliferation of neural progenitor cells during development. Basal progenitors, and in particular basal radial glial cells, are thought to have a key role in the increased generation of neurons that constitutes a foundation of neocortex expansion. Recent studies have identified primate-specific and human-specific genes and changes in gene expression that promote increased proliferative capacity of cortical progenitors. In many cases, the cell biological basis underlying this increase has been uncovered. Model systems such as mouse, ferret, nonhuman primates, and cerebral organoids have been used to establish the relevance of these genes for neocortex expansion.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex

The mammalian cerebral cortex comprises six layers of neurons. Cortical progenitors in the ventricular zone generate neurons specific to each layer through successive cell divisions. Neurons of layer VI are generated at an early stage, whereas later-born neurons occupy progressively upper layers. The underlying molecular mechanisms of neurogenesis, however, are relatively unknown. In this study, we devised a system where the Notch pathway was activated spatiotemporally in the cortex by in vivo electroporation and Cre-mediated DNA recombination. Electroporation at E13.5 transferred DNA to early progenitors that gave rise to neurons of both low and upper layers. Forced expression of a constitutively active form of Notch (caNotch) at E13.5 inhibited progenitors from generating neurons and kept progenitors as proliferating radial glial cells. After subsequent transfection at E15.5 of a Cre expression vector to remove caNotch, double-transfected cells, in which caNotch was excised, migrated into the cortical plate and differentiated into neurons specific to upper layers. Bromodeoxyuridine-labeling experiments showed that the neurons were born after Cre transfection. These results indicate that cortical progenitors that had been temporarily subjected to Notch activation at an early stage generated neurons at later stages, but that the generation of low-layer neurons was skipped. Moreover, the double-transfected cells gave rise to upper-layer neurons, even after their transplantation into the E13.5 brain, indicating that the developmental state of progenitors is not halted by caNotch activity.     

Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division

Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell (RGPC) neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture.     

How to keep proliferative neural stem/progenitor cells: A critical role of asymmetric inheritance of cyclin D2

It has long been argued that cell cycle regulators such as cyclins, cyclin-dependent kinases and their inhibitors affect the fate of neuronal progenitor cells. Recently, we identified that cyclin D2, which localizes at the basal tip of the radial glial cell (i.e., the neural progenitor in the developing neocortex), functions to give differential cell fates to its daughter cells just after cell division. This basally biased localization is due to transportation of cyclin D2 mRNA via its unique cis-regulatory sequence and local translation into cyclin D2 protein at the basal endfoot. During division of the neural progenitor cells, cyclin D2 protein is inherited by the daughter cell that retain the basal process, resulting in asymmetric distribution of cyclin D2 protein between the two daughter cells. Cyclin D2 is similarly localized in the human fetal cortical primordium, suggesting a common mechanism for the maintenance of neural progenitors and a possible scenario in evolution of primate brains. Here we introduce our recent findings and discuss how cyclin D2 functions in mammalian brain development and evolution.     

How to keep proliferative neural stem/progenitor cells

It has long been argued that cell cycle regulators such as cyclins, cyclin-dependent kinases and their inhibitors affect the fate of neuronal progenitor cells. Recently, we identified that cyclin D2, which localizes at the basal tip of the radial glial cell (i.e., the neural progenitor in the developing neocortex), functions to give differential cell fates to its daughter cells just after cell division. This basally biased localization is due to transportation of cyclin D2 mRNA via its unique cis-regulatory sequence and local translation into cyclin D2 protein at the basal endfoot. During division of the neural progenitor cells, cyclin D2 protein is inherited by the daughter cell that retain the basal process, resulting in asymmetric distribution of cyclin D2 protein between the two daughter cells. Cyclin D2 is similarly localized in the human fetal cortical primordium, suggesting a common mechanism for the maintenance of neural progenitors and a possible scenario in evolution of primate brains. Here we introduce our recent findings and discuss how cyclin D2 functions in mammalian brain development and evolution.     

Visual activity regulates neural progenitor cells in developing xenopus CNS through musashi1

Regulation of progenitor cell fate determines the numbers of neurons in the developing brain. While proliferation of neural progenitors predominates during early central nervous system (CNS) development, progenitor cell fate shifts toward differentiation as CNS circuits develop, suggesting that signals from developing circuits may regulate proliferation and differentiation. We tested whether activity regulates neurogenesis in?vivo in the developing visual system of Xenopus tadpoles. Both cell proliferation and the number of musashi1-immunoreactive progenitors in the optic tectum decrease as visual system connections become stronger. Visual deprivation for 2?days increased proliferation of musashi1-immunoreactive radial glial progenitors, while visual experience increased neuronal differentiation. Morpholino-mediated knockdown and overexpression of musashi1 indicate that musashi1 is necessary and sufficient for neural progenitor proliferation in the CNS. These data demonstrate a mechanism by which increased brain activity in developing circuits decreases cell proliferation and increases neuronal differentiation through the downregulation of musashi1 in response to circuit activity.     

In vivo fate analysis reveals the multipotent and self-renewal features of embryonic AspM expressing cells

Radial Glia (RG) cells constitute the major population of neural progenitors of the mouse developing brain. These cells are located in the ventricular zone (VZ) of the cerebral cortex and during neurogenesis they support the generation of cortical neurons. Later on, during brain maturation, RG cells give raise to glial cells and supply the adult mouse brain of Neural Stem Cells (NSC). Here we used a novel transgenic mouse line expressing the CreER(T2) under the control of AspM promoter to monitor the progeny of an early cohort of RG cells during neurogenesis and in the post natal brain. Long term fate mapping experiments demonstrated that AspM-expressing RG cells are multi-potent, as they can generate neurons, astrocytes and oligodendrocytes of the adult mouse brain. Furthermore, AspM descendants give also rise to proliferating progenitors in germinal niches of both developing and post natal brains. In the latter--i.e. the Sub Ventricular Zone--AspM descendants acquired several feature of neural stem cells, including the capability to generate neurospheres in vitro. We also performed the selective killing of these early progenitors by using a Nestin-GFP(flox)-TK allele. The forebrain specific loss of early AspM expressing cells caused the elimination of most of the proliferating cells of brain, a severe derangement of the ventricular zone architecture, and the impairment of the cortical lamination. We further demonstrated that AspM is expressed by proliferating cells of the adult mouse SVZ that can generate neuroblasts fated to become olfactory bulb neurons.     

EphB receptors couple dendritic filopodia motility to synapse formation

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 expression of aristaless-related homeobox in neural progenitors of gyrencephalic carnivore ferrets

Aristaless-related homeobox (ARX) has important functions in the development of various organs including the brain. Mutations of the human ARX gene have been associated with malformations of the cerebral cortex such as microcephaly and lissencephaly. Although the expression patterns of ARX in the lissencephalic cerebral cortex of mice have been intensively investigated, those in expanded gyrencephalic brains remained unclear. Here, we show the expression patterns of ARX in the developing cerebral cortex of gyrencephalic carnivore ferrets. We found that ARX was expressed not only in intermediate progenitor (IP) cells but also in outer radial glial (oRG) cells, which are neural progenitors preferentially observed in the gyrencephalic cerebral cortex. We found that the majority of ARX-positive oRG cells expressed the proliferating cell marker Ki-67. These results may indicate that ARX in oRG cells mediates the expansion of the gyrencephalic cerebral cortex during development and evolution.