The role of adherens junctions in the developing neocortex

The disproportional enlargement of the neocortex through evolution has been instrumental in the success of vertebrates, in particular mammals. The neocortex is a multilayered sheet of neurons generated from a simple proliferative neuroepithelium through a myriad of mechanisms with substantial evolutionary conservation. This developing neuroepithelium is populated by progenitors that can generate additional progenitors as well as post-mitotic neurons. Subtle alterations in the production of progenitors vs. differentiated cells during development can result in dramatic differences in neocortical size. This review article will examine how cadherin adhesion proteins, in particular α-catenin and N-cadherin, function in regulating the neural progenitor microenvironment, cell proliferation, and differentiation in cortical development.     

The microcephaly gene Donson is essential for progenitors of cortical glutamatergic and GABAergic neurons

Biallelic mutations in DONSON, an essential gene encoding for a replication fork protection factor, were linked to skeletal abnormalities and microcephaly. To better understand DONSON function in corticogenesis, we characterized Donson expression and consequences of conditional Donson deletion in the mouse telencephalon. Donson was widely expressed in the proliferation and differentiation zones of the embryonic dorsal and ventral telencephalon, which was followed by a postnatal expression decrease. Emx1-Cre-mediated Donson deletion in progenitors of cortical glutamatergic neurons caused extensive apoptosis in the early dorsomedial neuroepithelium, thus preventing formation of the neocortex and hippocampus. At the place of the missing lateral neocortex, these mutants exhibited a dorsal extension of an early-generated paleocortex. Targeting cortical neurons at the intermediate progenitor stage using Tbr2-Cre evoked no apparent malformations, whereas Nkx2.1-Cre-mediated Donson deletion in subpallial progenitors ablated 75% of Nkx2.1-derived cortical GABAergic neurons. Thus, the early telencephalic neuroepithelium depends critically on Donson function. Our findings help explain why the neocortex is most severely affected in individuals with DONSON mutations and suggest that DONSON-dependent microcephaly might be associated with so far unrecognized defects in cortical GABAergic neurons. Targeting Donson using an appropriate recombinase is proposed as a feasible strategy to ablate proliferating and nascent cells in experimental research.     

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.     

In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.     

Cortical development and asymmetric cell divisions

The development of the mammalian neocortex involves rounds of symmetric and asymmetric cell division of neural progenitors to fulfill needs of both self-renewal of progenitors and production of differe...     

Neuronal subtype specification in the cerebral cortex

In recent years, tremendous progress has been made in understanding the mechanisms underlying the specification of projection neurons within the mammalian neocortex. New experimental approaches have made it possible to identify progenitors and study the lineage relationships of different neocortical projection neurons. An expanding set of genes with layer and neuronal subtype specificity have been identified within the neocortex, and their function during projection neuron development is starting to be elucidated. Here, we assess recent data regarding the nature of neocortical progenitors, review the roles of individual genes in projection neuron specification and discuss the implications for progenitor plasticity.     

Widespread tangential dispersion and extensive cell death during early neurogenesis in the mouse neocortex

The development of the mammalian neocortex requires radial and tangential migration of cells. Radial migration of differentiated neurons from the ventricular zone (VZ) is well established. It is hypothesised that an earlier phase of tangential migration of mitotically active cells lays down a widespread periodically spaced set of progenitors that generate radial arrays of postmitotic neurons. We use a transgenic cell lineage marker to label and observe the behaviour of progenitors before and during the early stages of neurogenesis. Using optical projection tomography (OPT), we show that individual progenitor cells generate many radially arrayed columns of periodically spaced cells. Column positions indicate the paths taken by these progenitor cells as they migrate, often over long distances, through the proliferative zone. Clonally related cells can be distributed in both hemispheres, suggesting progenitor cells cross the midline in the anterior neural plate. We observe a dramatic and rapid decline in the number of labelled clones after E13.5, indicating that there is extensive cell death at this time.     

Tangential migration and proliferation of intermediate progenitors of GABAergic neurons in the mouse telencephalon

In the embryonic neocortex, neuronal precursors are generated in the ventricular zone (VZ) and accumulate in the cortical plate. Recently, the subventricular zone (SVZ) of the embryonic neocortex was recognized as an additional neurogenic site for both principal excitatory neurons and GABAergic inhibitory neurons. To gain insight into the neurogenesis of GABAergic neurons in the SVZ, we investigated the characteristics of intermediate progenitors of GABAergic neurons (IPGNs) in mouse neocortex by immunohistochemistry, immunocytochemistry, single-cell RT-PCR and single-cell array analysis. IPGNs were identified by their expression of some neuronal and cell cycle markers. Moreover, we investigated the origins of the neocortical IPGNs by Cre-loxP fate mapping in transgenic mice and the transduction of part of the telencephalic VZ by Cre-reporter plasmids, and found them in the medial and lateral ganglionic eminence. Therefore, they must migrate tangentially within the telencephalon to reach the neocortex. Cell-lineage analysis by simple-retrovirus transduction revealed that the neocortical IPGNs self-renew and give rise to a small number of neocortical GABAergic neurons and to a large number of granule and periglomerular cells in the olfactory bulb. IPGNs are maintained in the neocortex and may act as progenitors for adult neurogenesis.     

An in vitro model of human neocortical development using pluripotent stem cells: cocaine-induced cytoarchitectural alterations

Neocortical development involves ordered specification of forebrain cortical progenitors to various neuronal subtypes, ultimately forming the layered cortical structure. Modeling of this process using human pluripotent stem cells (hPSCs) would enable mechanistic studies of human neocortical development, while providing new avenues for exploration of developmental neocortical abnormalities. Here, we show that preserving hPSCs aggregates – allowing embryoid body formation – while adding basic fibroblast growth factor (bFGF) during neuroepithelial development generates neural rosettes showing dorsal forebrain identity, including Mash1⁺ dorsal telencephalic GABAergic progenitors. Structures that mirrored the organization of the cerebral cortex formed after rosettes were seeded and cultured for 3 weeks in the presence of FGF18, BDNF and NT3. Neurons migrated along radial glia scaffolding, with deep-layer CTIP2⁺ cortical neurons appearing after 1 week and upper-layer SATB2⁺ cortical neurons forming during the second and third weeks. At the end of differentiation, these structures contained both glutamatergic and GABAergic neurons, with glutamatergic neurons being most abundant. Thus, this differentiation protocol generated an hPSC-based model that exhibits temporal patterning and a neuronal subtype ratio similar to that of the developing human neocortex. This model was used to examine the effects of cocaine during neocorticogenesis. Cocaine caused premature neuronal differentiation and enhanced neurogenesis of various cortical neuronal subtypes. These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine. This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.KEY WORDS: Neocortical development, Dorsal forebrain model, hPSCs, Cocaine, Premature neuronal differentiation     

G protein betagamma subunits and AGS3 control spindle orientation and asymmetric cell fate of cerebral cortical progenitors

Neurons in the developing mammalian brain are generated from progenitor cells in the proliferative ventricular zone, and control of progenitor division is essential to produce the correct number of neurons during neurogenesis. Here we establish that Gbetagamma subunits of heterotrimeric G proteins are required for proper mitotic-spindle orientation of neural progenitors in the developing neocortex. Interfering with Gbetagamma function in progenitors causes a shift in spindle orientation from apical-basal divisions to planar divisions. This results in hyperdifferentiation of progenitors into neurons as a consequence of both daughter cells adopting a neural fate instead of the normal asymmetric cell fates. Silencing AGS3, a nonreceptor activator of Gbetagamma, results in defects similar to the impairment of Gbetagamma, providing evidence that AGS3-Gbetagamma signaling in progenitors regulates apical-basal division and asymmetric cell-fate decisions. Furthermore, our observations indicate that the cell-fate decision of daughter cells is coupled to mitotic-spindle orientation in progenitors.     

Cortical progenitor expansion, self-renewal and neurogenesis-a polarized perspective

Neural stem and progenitor cells giving rise to neurons in developing mammalian neocortex fall into two principal classes with regard to location of mitosis-apical and basal, and into three principal classes in terms of cell polarity during mitosis-bipolar, monopolar, and nonpolar. Insight has been gained into how inheritance of polarized, apical and basal, cell constituents is related to symmetric versus asymmetric divisions of these progenitors, and how this inheritance is linked to their expansion, self-renewal, and neurogenesis. Retention and inheritance of the basal process emerge as key for self-renewal, notably for the monopolar progenitors of prospective gyrencephalic neocortex that undergo asymmetric mitoses at basal locations. The resulting expansion of the neocortex during evolution is proposed to be associated with an increased cone-shape of radial units.     

Discs large 5 is required for polarization of citron kinase in mitotic neural precursors

Citron kinase (CitK), a protein essential to neurogenic cell division in the central nervous system, is highly polarized in neural progenitors. The mechanisms that polarize CitK to cellular domains that line the ventricular surface of neuroepithelium are currently not known. Here we report that Discs large 5 (Dlg5), a member of the MAGUK family, is an interactor of CitK required for CitK polarization. The CitK-Dlg5 interaction was first revealed in a protein array screen of proteins containing PDZ domains, and then subsequently confirmed by co-immunoprecipitation. Moreover, in Dlg5^-/- mice CitK fails to polarize in mitotic neuronal precursors. In addition, the total number of mitotic progenitors and the ratio of ventricular to abventricular mitotic progenitors in developing neocortex are significantly decreased in Dlg5^-/- embryos. Dlg5 is therefore required to maintain the polarization of a protein essential to neurogenic cytokinesis, and plays a role in localizing cell divisions to the surface of the lateral ventricles in embryonic brain.     

The temporal sequence of the mammalian neocortical neurogenetic program drives mediolateral pattern in the chick pallium

The six-layered neocortex permits complex information processing in all mammalian species. Because its homologous region (the pallium) in nonmammalian amniotes has a different architecture, the ability of neocortical progenitors to generate an orderly sequence of distinct cell types was thought to have arisen in the mammalian lineage. This study, however, shows that layer-specific neuron subtypes do exist in the chick pallium. Deep- and upper-layer neurons are not layered but are segregated in distinct mediolateral domains in vivo. Surprisingly, cultured chick neural progenitors produce multiple layer-specific neuronal subtypes in the same chronological sequence as seen in mammals. These results suggest that the temporal sequence of the neocortical neurogenetic program was already inherent in the last common ancestor of mammals and birds and that mammals use this conserved program to generate a uniformly layered neocortex, whereas birds impose spatial constraints on the sequence to pattern the pallium.