Basal Radial Glial Cells Research Articles

Biallelic null variants in PNPLA8 cause microcephaly by reducing the number of basal radial glia.

Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development.

Dynamic changes in mitochondrial localization in human neocortical basal radial glial cells during cell cycle.

Mitochondria play critical roles in neural stem/progenitor cell proliferation and fate decisions. The subcellular localization of mitochondria in neural stem/progenitor cells during mitosis potentially influences the distribution of mitochondria to the daughter cells and thus their fates. Therefore, understanding the spatial dynamics of mitochondria provides important knowledge about brain development. In this study, we analyzed the subcellular localization of mitochondria in the fetal human neocortex with a particular focus on the basal radial glial cells (bRGCs), a neural stem/progenitor cell subtype attributed to the evolutionary expansion of the human neocortex. During interphase, bRGCs exhibit a polarized localization of mitochondria that is localized at the base of the process or the proximal part of the process. Thereafter, mitochondria in bRGCs at metaphase show unpolarized distribution in which the mitochondria are randomly localized in the cytoplasm. During anaphase and telophase, mitochondria are still localized evenly, but mainly in the periphery of the cytoplasm. Mitochondria start to accumulate at the cleavage furrow during cytokinesis. These results suggest that the mitochondrial localization in bRGCs is tightly regulated during the cell cycle, which may ensure the proper distribution of mitochondria to the daughter cells and, thus in turn, influence their fates.

A cell fate decision map reveals abundant direct neurogenesis bypassing intermediate progenitors in the human developing neocortex

The human neocortex has undergone strong evolutionary expansion, largely due to an increased progenitor population, the basal radial glial cells. These cells are responsible for the production of a diversity of cell types, but the successive cell fate decisions taken by individual progenitors remain unknown. Here we developed a semi-automated live/fixed correlative imaging method to map basal radial glial cell division modes in early fetal tissue and cerebral organoids. Through the live analysis of hundreds of dividing progenitors, we show that basal radial glial cells undergo abundant symmetric amplifying divisions, and frequent self-consuming direct neurogenic divisions, bypassing intermediate progenitors. These direct neurogenic divisions are more abundant in the upper part of the subventricular zone. We furthermore demonstrate asymmetric Notch activation in the self-renewing daughter cells, independently of basal fibre inheritance. Our results reveal a remarkable conservation of fate decisions in cerebral organoids, supporting their value as models of early human neurogenesis.

FOXP1 orchestrates neurogenesis in human cortical basal radial glial cells.

During cortical development, human basal radial glial cells (bRGCs) are highly capable of sustained self-renewal and neurogenesis. Selective pressures on this cell type may have contributed to the evolution of the human neocortex, leading to an increase in cortical size. bRGCs have enriched expression for Forkhead Box P1 (FOXP1), a transcription factor implicated in neurodevelopmental disorders (NDDs) such as autism spectrum disorder. However, the cell type-specific roles of FOXP1 in bRGCs during cortical development remain unexplored. Here, we examine the requirement for FOXP1 gene expression regulation underlying the production of bRGCs using human brain organoids. We examine a developmental time point when FOXP1 expression is highest in the cortical progenitors, and the bRGCs, in particular, begin to actively produce neurons. With the loss of FOXP1, we show a reduction in the number of bRGCs, as well as reduced proliferation and differentiation of the remaining bRGCs, all of which lead to reduced numbers of excitatory cortical neurons over time. Using single-nuclei RNA sequencing and cell trajectory analysis, we uncover a role for FOXP1 in directing cortical progenitor proliferation and differentiation by regulating key signaling pathways related to neurogenesis and NDDs. Together, these results demonstrate that FOXP1 regulates human-specific features in early cortical development.

Ethanol exposure disrupted the formation of radial glial processes and impaired the generation and migration of outer radial glial cells in forebrain organoids derived from human embryonic stem cells

Radial glial cells (RGCs) play a pivotal role in cerebral cortical development by functioning as a source of new neurons and by supporting the migration of newborn neurons. These functions are primarily dependent on the apical-basolateral structures of radial glial processes. This study aims to investigate the effects of ethanol exposure on the development of radial glial processes and the generation, migration, and transformation of outer radial glial cells (oRGCs). For this purpose, forebrain organoids were developed from human embryonic stem cells. These forebrain organoids contain abundant neural progenitor cells (SOX2+), express high levels of neural epithelial markers β-catenin and PKCλ, and dorsal forebrain marker PAX6, and display well-organized cortical architectures containing abundant apical and basal RGCs, intermediate progenitors (IPCs), and neurons. Exposure of forebrain organoids to ethanol resulted in a significant increase in apoptosis in Nestin-positive radial glial cells. Ethanol exposure also remarkably decreased the levels of radial glial process-associated proteins, including Nestin, GFAP, and Vimentin, in radial glial cells and distinctly impaired the integrity and morphologies of radial glial processes. In addition, the ethanol-induced impairment of the radial glial processes is associated with decreased migration and proliferation of radial glial cells, reduction in the generation of HOPX+ oRGCs, and the accelerated transformation of oRGCs into astrocytes. These results demonstrate that ethanol exposure can disrupt cerebral cortex development by impairing the formation of radial glial processes and the generation, migration, and transformation of oRGCs.

CAMSAPs organize an acentrosomal microtubule network from basal varicosities in radial glial cells.

Neurons of the neocortex are generated by stem cells called radial glial cells. These polarized cells extend a short apical process toward the ventricular surface and a long basal fiber that acts as a scaffold for neuronal migration. How the microtubule cytoskeleton is organized in these cells to support long-range transport is unknown. Using subcellular live imaging within brain tissue, we show that microtubules in the apical process uniformly emanate for the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, reminiscent of the mammalian dendrite. We identify acentrosomal microtubule organizing centers localized in varicosities of the basal fiber. CAMSAP family members accumulate in these varicosities, where they control microtubule growth. Double knockdown of CAMSAP1 and 2 leads to a destabilization of the entire basal process. Finally, using live imaging of human fetal cortex, we reveal that this organization is conserved in basal radial glial cells, a related progenitor cell population associated with human brain size expansion.

Prenatal ethanol exposure impairs the formation of radial glial fibers and promotes the transformation of GFAPδ‑positive radial glial cells into astrocytes.

During embryonic cortical development, radial glial cells (RGCs) are the major source of neurons, and these also serve as a supportive scaffold to guide neuronal migration. Similar to Vimentin, glial fibrillary acidic protein (GFAP) is one of the major intermediate filament proteins present in glial cells. Previous studies confirmed that prenatal ethanol exposure (PEE) significantly affected the levels of GFAP and increased the disassembly of radial glial fibers. GFAPδ is a variant of GFAP that is specifically expressed in RGCs; however, to the best of our knowledge, there are no reports regarding how PEE influences its expression during cortical development. In the present study, the effects of PEE on the expression and distribution of GFAPδ during early cortical development were assessed. It was found that PEE significantly decreased the expression levels of GFAP and GFAPδ. Using double immunostaining, GFAPδ was identified to be specifically expressed in apical and basal RGCs, and was co-localized with other intermediate filament proteins, such as GFAP, Nestin and Vimentin. Additionally, PEE significantly affected the morphology of radial glial fibers and altered the behavior of RGCs. The loss of GFAPδ accelerated the transformation of RGCs into astrocytes. Using co-immunostaining with Ki67 or phospho-histone H3, GFAPδ+ cells were observed to be proliferative or mitotic cells, and ethanol treatment significantly decreased the proliferative or mitotic activities of GFAPδ+ RGCs. Taken together, the results suggested that PEE altered the expression patterns of GFAPδ and impaired the development of radial glial fibers and RGC behavior. The results of the present study provided evidence that GFAPδ may be a promising target to rescue the damage induced by PEE.

Cell-Scale Modeling to Probe Mechanobiology during Early Cortical Development

Background/Objective: During early cerebral cortex development, neurons form from proliferative glial cells near the ventricular (apical) surface, then migrate along radial glial scaffolds to the cortical surface. In species with wrinkled brains, the presence of basal radial glial cells (bRGCs), radial glial cells which have detached from the ventricular surface, is correlated to the process of gyrification. While mechanical forces are also involved in gyrus creation, the link between the mechanical and biological aspects of this process remains unelucidated. In this study, we hypothesized that radial tension may lead to the production of gyri via the intermediary creation of bRGCs. 
 Methods: To test this hypothesis, the cell-level modeling software CX3D was used to simulate a system in which radial tension acts on radial glial cells (RGCs), facilitating the semi-stochastic production of bRGCs during the process of neocortex development. The outcome of this model was contrasted with a control case in which bRGCs were not allowed to form, and the two models were compared based upon the presence of neurons on the basal surface. 
 Results: The production of bRGCs via tension corresponded to a significant increase in the presence of neurons on the pial surface, even if the total number of glial cells—and thus total number of neurons generated—remained constant. Additionally, the likelihood of neurons moving more basally was found to be significantly greater in the presence of bRGCs. 
 Conclusion and Potential Impact: These results were interpreted to be indications of early gyrus formation. Thus, this study showed that bRGCs—and, ultimately, gyri—may arise from mechanical tension, indicating a possible link between the biological and mechanical explanations of gyrus formation. By providing an alternative lens through which to understand cortical folding, this may have implications for future lines of inquiry, which may expand our understanding of neuro-pathologies associated with misfolding, such as autism and epilepsy.

Genes and Mechanisms Involved in the Generation and Amplification of Basal Radial Glial Cells.

The development of the cerebral cortex relies on different types of progenitor cell. Among them, the recently described basal radial glial cell (bRG) is suggested to be of critical importance for the development of the brain in gyrencephalic species. These cells are highly numerous in primate and ferret brains, compared to lissencephalic species such as the mouse in which they are few in number. Their somata are located in basal subventricular zones in gyrencephalic brains and they generally possess a basal process extending to the pial surface. They sometimes also have an apical process directed toward the ventricular surface, similar to apical radial glial cells (aRGs) from which they are derived, and whose somata are found more apically in the ventricular zone. bRGs share similarities with aRGs in terms of gene expression (SOX2, PAX6, and NESTIN), whilst also expressing a range of more specific genes (such as HOPX). In primate brains, bRGs can divide multiple times, self-renewing and/or generating intermediate progenitors and neurons. They display a highly specific cytokinesis behavior termed mitotic somal translocation. We focus here on recently identified molecular mechanisms associated with the generation and amplification of bRGs, including bRG-like cells in the rodent. These include signaling pathways such as the FGF-MAPK cascade, SHH, PTEN/AKT, PDGF pathways, and proteins such as INSM, GPSM2, ASPM, TRNP1, ARHGAP11B, PAX6, and HIF1α. A number of these proteins were identified through transcriptome comparisons in human aRGs vs. bRGs, and validated by modifying their activities or expression levels in the mouse. This latter experiment often revealed enhanced bRG-like cell production, even in some cases generating folds (gyri) on the surface of the mouse cortex. We compare the features of the identified cells and methods used to characterize them in each model. These important data converge to indicate pathways essential for the production and expansion of bRGs, which may help us understand cortical development in health and disease.

A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.

A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.

Neural progenitor cells and their role in the development and evolutionary expansion of the neocortex.

The evolutionary expansion of the mammalian brain, notably the neocortex, provides a platform for the higher cognitive abilities that characterize humans. Cortical expansion is accompanied by increased folding of the pial surface, which gives rise to a gyrencephalic (folded) rather than lissencephalic (unfolded) neocortex. This expansion reflects the prolonged and increased proliferation of neural stem and progenitor cells (NPCs). Distinct classes of NPCs can be distinguished based on either cell biological criteria (apical progenitors [APs], basal progenitors [BPs]) or lineage (primary progenitors and secondary progenitors). Cortical expansion in development and evolution is linked to an increased abundance and proliferative capacity of BPs, notably basal radial glial cells, a recently characterized type of secondary progenitor derived from apical radial glial cells, the primary progenitors. To gain insight into the molecular basis underlying the prolonged and increased proliferation of NPCs and in particular BPs, comparative genomic and transcriptomic approaches, mostly for human versus mouse, have been employed and applied to specific NPC types and subpopulations. These have revealed two principal sets of molecular changes. One concerns differences in the expression of common genes between species with different degrees of cortical expansion. The other comprises human-specific genes or genomic regulatory sequences. Various systems that allow functional testing of these genomic and gene expression differences between species have emerged, including transient and stable transgenesis, genome editing, cerebral organoids, and organotypic slice cultures. These provide future avenues for uncovering the molecular basis of cortical expansion. WIREs Dev Biol 2017, 6:e256. doi: 10.1002/wdev.256 For further resources related to this article, please visit the WIREs website.

Apical Polarization of SVCT2 in Apical Radial Glial Cells and Progenitors During Brain Development.

During brain development, radial glial (RG) cells and the different progenitor subtypes are characterized by their bipolar morphology that includes an ovoid cell body and one or two radial processes that span across the developing cerebral wall. Different cells transport the reduced form of vitamin C, ascorbic acid (AA), using sodium-dependent ascorbic acid cotransporters (SVCT1 or SVCT2). SVCT2 is mainly expressed in the nervous system (CNS); however, its localization in the central nervous system during embryonic development along with the mechanism by which RG take up vitamin C and its intracellular effects is unknown. Thus, we sought to determine the expression and localization of SVCT2 during CNS development. SVCT2 is preferentially localized in the RG body at the ventricular edge of the cortex during the neurogenic stage (E12 to E17). The localization of SVCT2 overexpressed by in utero electroporation of E14 embryos is consistent with ventricular polarization. A similar distribution pattern was observed in human brain tissue sections at 9weeks of gestation; however, SVCT2 immunoreaction was also detected in the inner and outer subventricular zone (SVZ). Finally, we used C17.2 neural stem cell line, J1ES cells and primary cell cultures derived from the brain cortex to analyze functional SVCT2 activity, AA effects in progenitor cells bipolar morphology, and SVCT2 expression levels in different culture conditions. Our results indicate that basal RG cells and apical intermediate and subapical progenitors are the main cell types expressing SVCT2 in the lissencephalic brain. SVCT2 was mainly detected in the apical region of the ventricular zone cells, contacting thecerebrospinal fluid. In gyrencephalic brains, SVCT2 was also detected in progenitor cells located in the inner and outer SVZ. Finally, we defined that AA has a strong radializing (bipolar morphology) effect in progenitor cells in culture and the differentiation condition modulates SVCT2 expression.

The evolution of basal progenitors in the developing non-mammalian brain.

The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2+ intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2+ cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution.

Trnp1 Regulates Expansion and Folding of the Mammalian Cerebral Cortex by Control of Radial Glial Fate

Evolution of the mammalian brain encompassed a remarkable increase in size of the cerebral cortex, which includes tangential and radial expansion. However, the mechanisms underlying these key features are still largely unknown. Here, we identified the DNA-associated protein Trnp1 as a regulator of cerebral cortex expansion in both of these dimensions. Gain- and loss-of-function experiments in the mouse cerebral cortex invivo demonstrate that high Trnp1 levels promote neural stem cell self-renewal and tangential expansion. In contrast, lower levels promote radial expansion, with a potent increase of the number of intermediate progenitors and basal radial glial cells leading to folding of the otherwise smooth murine cerebral cortex. Remarkably, TRNP1 expression levels exhibit regional differences in the cerebral cortex of human fetuses, anticipating radial or tangential expansion. Thus, the dynamic regulation of Trnp1 is critical to control tangential and radial expansion of the cerebral cortex in mammals.

Screening for candidate genes involved in the production of mouse subventricular zone proliferative cells and an estimation of their changes in evolutionary pressure during primate evolution

During neocortical development, excitatory neurons are produced from apical progenitors in the ventricular zone (VZ) or from dividing cells in the subventricular zone (SVZ). We previously reported that the direct progenies of VZ cells in mice slowly exit the VZ and accumulate just above the VZ (lower SVZ) as multipolar migrating neurons, whereas subsequently dividing cells in the SVZ exit the VZ earlier than the former and become widely distributed in the SVZ. These two populations are named the slowly exiting population (SEP) and the rapidly exiting population (REP), respectively. In mice, REP cells include basal progenitors as the major population and are characterized by a long ascending process; their morphology resembles that of basal radial glial cells (bRGs), which have been observed in the inner and outer SVZ in primates. The dramatic increase in the number of bRGs in primates, especially in humans, is thought to underlie the acquisition of a huge cortex during evolution. We previously reported that the REP/SEP production rate in the lateral cortical VZ is higher than that in the dorsomedial VZ in mice. To search for molecules responsible for the higher REP production in the lateral cortical VZ, we conducted microarray analyses and identified genes that were differentially expressed between the lateral and medial VZs in mice. These genes were considered to be among the candidates responsible for the regulation of the REP/SEP production rate. To investigate the selection pressures during primate evolution on these candidate genes, we estimated the synonymous vs. non-synonymous base substitution rates. As a result, the negative selection pressures on the Megf11, Dmrt3, and Cntn3 genes were found to be significantly weaker in primates than in non-primates, while those on Jag1, Ntrk2, and Pmp22 were stronger. Candidate molecules responsible for primate cortical expansion through an increase in bRGs may be included among these genes.