Embryonic mouse medial neocortex as a model system for studying the radial glial scaffold in fetal human neocortex.

Neocortex is the evolutionarily newest region in the brain, and is a structure with diversified size and morphology among mammalian species. Humans have the biggest neocortex compared to the body size, and their neocortex has many foldings, that is, gyri and sulci. Despite the recent methodological advances in in vitro models such as cerebral organoids, mice have been continuously used as a model system for studying human neocortical development because of the accessibility and practicality of in vivo gene manipulation. The commonly studied neocortical region, the lateral neocortex, generally recapitulates the developmental process of the human neocortex, however, there are several important factors missing in the lateral neocortex. First, basal (outer) radial glia (bRG), which are the main cell type providing the radial scaffold to the migrating neurons in the fetal human neocortex, are very few in the mouse lateral neocortex, thus the radial glial scaffold is different from the fetal human neocortex. Second, as a consequence of the difference in the radial glial scaffold, migrating neurons might exhibit different migratory behavior and thus distribution. To overcome those problems, we propose the mouse medial neocortex, where we have earlier revealed an abundance of bRG similar to the fetal human neocortex, as an alternative model system. We found that similar to the fetal human neocortex, the radial glial scaffold, neuronal migration and neuronal distribution are tangentially scattered in the mouse medial neocortex. Taken together, the embryonic mouse medial neocortex could be a suitable and accessible in vivo model system to study human neocortical development and its pathogenesis.

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.

Transcriptional Regulators and Human-Specific/Primate-Specific Genes in Neocortical Neurogenesis.

During development, starting from a pool of pluripotent stem cells, tissue-specific genetic programs help to shape and develop functional organs. To understand the development of an organ and its disorders, it is important to understand the spatio-temporal dynamics of the gene expression profiles that occur during its development. Modifications in existing genes, the de-novo appearance of new genes, or, occasionally, even the loss of genes, can greatly affect the gene expression profile of any given tissue and contribute to the evolution of organs or of parts of organs. The neocortex is evolutionarily the most recent part of the brain, it is unique to mammals, and is the seat of our higher cognitive abilities. Progenitors that give rise to this tissue undergo sequential waves of differentiation to produce the complete sets of neurons and glial cells that make up a functional neocortex. We will review herein our understanding of the transcriptional regulators that control the neural precursor cells (NPCs) during the generation of the most abundant class of neocortical neurons, the glutametergic neurons. In addition, we will discuss the roles of recently-identified human- and primate-specific genes in promoting neurogenesis, leading to neocortical expansion.

PUF-8 negatively regulates RAS/MAPK signalling to promote differentiation of C. elegans germ cells.

Signals that promote germ cell self-renewal by preventing premature meiotic entry are well understood. However, signals that control mitotic proliferation to promote meiotic differentiation have not been well characterized. In Caenorhabditis elegans, GLP-1 Notch signalling promotes the proliferative fate by preventing premature meiotic entry. The germline niche cell, which is the source of the ligand for GLP-1, spatially restricts GLP-1 signalling and thus enables the germ cells that have moved away from the niche to enter meiosis. Here, we show that the suppression of RAS/MAP kinase signalling in the mitotic and meiotic-entry regions is essential for the regulation of the mitosis-meiosis switch by niche signalling. We provide evidence that the conserved PUF family RNA-binding protein PUF-8 and the RAS GAP protein GAP-3 function redundantly to suppress the LET-60 RAS in the mitotic and meiotic entry regions. Germ cells missing both PUF-8 and GAP-3 proliferate in an uncontrolled fashion and fail to undergo meiotic development. MPK-1, the MAP kinase downstream of the LET-60 RAS, is prematurely activated in these cells; downregulation of MPK-1 activation eliminates tumours and restores differentiation. Our results further reveal that PUF-8 negatively regulates LET-60 expression at a post-transcriptional step. LET-60 is misexpressed in the puf-8(-) mutant germlines and PUF-8 physically interacts with the let-60 3' UTR. Furthermore, PUF-8 suppresses let-60 3' UTR-mediated expression in the germ cells that are transitioning from the mitotic to meiotic fate. These results reveal that PUF-8-mediated inhibition of the RAS/MAPK pathway is essential for mitotic-to-meiotic fate transition.

Migration-Associated Transportome and Therapeutic Potential in Glioblastoma Multiforme (GBM).

GBM is a highly aggressive and very common malignant form of primary brain tumors in adults [...].

Progenitor-Based Cell Biological Aspects of Neocortex Development and Evolution.

During development, the decision of stem and progenitor cells to switch from proliferation to differentiation is of critical importance for the overall size of an organ. Too early a switch will deplete the stem/progenitor cell pool, and too late a switch will not generate the required differentiated cell types. With a focus on the developing neocortex, a six-layered structure constituting the major part of the cerebral cortex in mammals, we discuss here the cell biological features that are crucial to ensure the appropriate proliferation vs. differentiation decision in the neural progenitor cells. In the last two decades, the neural progenitor cells giving rise to the diverse types of neurons that function in the neocortex have been intensely investigated for their role in cortical expansion and gyrification. In this review, we will first describe these different progenitor types and their diversity. We will then review the various cell biological features associated with the cell fate decisions of these progenitor cells, with emphasis on the role of the radial processes emanating from these progenitor cells. We will also discuss the species-specific differences in these cell biological features that have allowed for the evolutionary expansion of the neocortex in humans. Finally, we will discuss the emerging role of cell cycle parameters in neocortical expansion.

Length of the Neurogenic Period-A Key Determinant for the Generation of Upper-Layer Neurons During Neocortex Development and Evolution.

The neocortex, a six-layer neuronal brain structure that arose during the evolution of, and is unique to, mammals, is the seat of higher order brain functions responsible for human cognitive abilities. Despite its recent evolutionary origin, it shows a striking variability in size and folding complexity even among closely related mammalian species. In most mammals, cortical neurogenesis occurs prenatally, and its length correlates with the length of gestation. The evolutionary expansion of the neocortex, notably in human, is associated with an increase in the number of neurons, particularly within its upper layers. Various mechanisms have been proposed and investigated to explain the evolutionary enlargement of the human neocortex, focussing in particular on changes pertaining to neural progenitor types and their division modes, driven in part by the emergence of human-specific genes with novel functions. These led to an amplification of the progenitor pool size, which affects the rate and timing of neuron production. In addition, in early theoretical studies, another mechanism of neocortex expansion was proposed-the lengthening of the neurogenic period. A critical role of neurogenic period length in determining neocortical neuron number was subsequently supported by mathematical modeling studies. Recently, we have provided experimental evidence in rodents directly supporting the mechanism of extending neurogenesis to specifically increase the number of upper-layer cortical neurons. Moreover, our study examined the relationship between cortical neurogenesis and gestation, linking the extension of the neurogenic period to the maternal environment. As the exact nature of factors promoting neurogenic period prolongation, as well as the generalization of this mechanism for evolutionary distinct lineages, remain elusive, the directions for future studies are outlined and discussed.

Generation of interspecies mouse-rat chimeric embryos by embryonic stem (ES) cell microinjection.

Interspecies chimerism is a useful tool to study interactions between cells of different genetic makeup in order to elucidate the mechanisms underlying non-cell-autonomous processes, including evolutionary events. However, generating interspecies chimeras with high efficiency and chimerism level remains challenging. Here, we describe a protocol for generating chimeras between mouse and rat. Donor embryonic stem cells of one species are microinjected into early embryos of the other species (recipient), which are implanted into host foster mothers of the recipient species. For complete details on the use and execution of this protocol, please refer to Stepien et al. (2020).

Serotonin Receptor 2A Activation Promotes Evolutionarily Relevant Basal Progenitor Proliferation in the Developing Neocortex.

Evolutionary expansion of the mammalian neocortex (Ncx) has been linked to increased abundance and proliferative capacity of basal progenitors (BPs) in the subventricular zone during development. BP proliferation is governed by both intrinsic and extrinsic signals, several of which have been identified. However, a role of neurotransmitters, a canonical class of extrinsic signaling molecules, in BP proliferation remains to be established. Here, we show that serotonin (5-HT), via its receptor HTR2A, promotes BP proliferation in an evolutionarily relevant manner. HTR2A is not expressed in embryonic mouse Ncx; accordingly, 5-HT does not increase mouse BP proliferation. However, ectopic HTR2A expression can increase mouse BP proliferation. Conversely, CRISPR/Cas9-mediated knockout of endogenous HTR2A in embryonic ferret Ncx reduces BP proliferation. Pharmacological activation of endogenous HTR2A in fetal human Ncx ex vivo increases BP proliferation via HER2/ERK signaling. Hence, 5-HT emerges as an important extrinsic pro-proliferative signal for BPs, which may have contributed to evolutionary Ncx expansion.

Lengthening Neurogenic Period during Neocortical Development Causes a Hallmark of Neocortex Expansion.

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.

Extracellular matrix-inducing Sox9 promotes both basal progenitor proliferation and gliogenesis in developing neocortex.

Neocortex expansion is largely based on the proliferative capacity of basal progenitors (BPs), which is increased by extracellular matrix (ECM) components via integrin signaling. Here we show that the transcription factor Sox9 drives expression of ECM components and that laminin 211 increases BP proliferation in embryonic mouse neocortex. We show that Sox9 is expressed in human and ferret BPs and is required for BP proliferation in embryonic ferret neocortex. Conditional Sox9 expression in the mouse BP lineage, where it normally is not expressed, increases BP proliferation, reduces Tbr2 levels and induces Olig2 expression, indicative of premature gliogenesis. Conditional Sox9 expression also results in cell-non-autonomous stimulation of BP proliferation followed by increased upper-layer neuron production. Our findings demonstrate that Sox9 exerts concerted effects on transcription, BP proliferation, neuron production, and neurogenic vs. gliogenic BP cell fate, suggesting that Sox9 may have contributed to promote neocortical expansion.

Human-Specific ARHGAP11B Acts in Mitochondria to Expand Neocortical Progenitors by Glutaminolysis.

The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion.

Primate neocortex development and evolution: Conserved versus evolved folding.

The neocortex, the seat of higher cognitive functions, exhibits a key feature across mammalian species-a highly variable degree of folding. Within the neocortex, two distinct subtypes of cortical areas can be distinguished, the isocortex and the proisocortex. Here, we have compared specific spatiotemporal aspects of folding between the proisocortex and the isocortex in 13 primates, including human, chimpanzee, and various Old World and New World monkeys. We find that folding at the boundaries of the dorsal isocortex and the proisocortex, which gives rise to the cingulate sulcus (CiS) and the lateral fissure (LF), is conserved across the primates studied and is therefore referred to as conserved folding. In contrast, the degree of folding within the dorsal isocortex exhibits huge variation across these primates, indicating that this folding, which gives rise to gyri and sulci, is subject to major changes during primate evolution. We therefore refer to the folding within the dorsal isocortex as evolved folding. Comparison of fetal neocortex development in long-tailed macaque and human reveals that the onset of conserved folding precedes the onset of evolved folding. Moreover, the analysis of infant human neocortex exhibiting lissencephaly, a developmental malformation thought to be mainly due to abnormal neuronal migration, shows that the evolved folding is perturbed more than the conserved folding. Taken together, our study presents a two-step model of folding that pertains to primate neocortex development and evolution. Specifically, our data imply that the conserved folding and the evolved folding constitute two distinct, sequential events.