Functional synergy of a human-specific and an ape-specific metabolic regulator in human neocortex development.

Metabolism has recently emerged as a major target of genes implicated in the evolutionary expansion of human neocortex. One such gene is the human-specific gene ARHGAP11B. During human neocortex development, ARHGAP11B increases the abundance of basal radial glia, key progenitors for neocortex expansion, by stimulating glutaminolysis (glutamine-to-glutamate-to-alpha-ketoglutarate) in mitochondria. Here we show that the ape-specific protein GLUD2 (glutamate dehydrogenase 2), which also operates in mitochondria and converts glutamate-to-αKG, enhances ARHGAP11B's ability to increase basal radial glia abundance. ARHGAP11B + GLUD2 double-transgenic bRG show increased production of aspartate, a metabolite essential for cell proliferation, from glutamate via alpha-ketoglutarate and the TCA cycle. Hence, during human evolution, a human-specific gene exploited the existence of another gene that emerged during ape evolution, to increase, via concerted changes in metabolism, progenitor abundance and neocortex size.

Response to Comment on "Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals".

Herai et al. discuss the known fact that a low percentage of modern humans who lack any overt phenotypes carry the ancestral TKTL1 allele. Our paper demonstrates that the amino acid substitution in TKTL1 increases neural progenitor cells and neurogenesis in the developing brain. It is another question if, and to what extent, this has consequences for the adult brain.

Comment on "Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals".

Pinson et al. (1) concluded that the modern human TKTL1 gene is responsible for an increased number of cortical neurons. We show that the "putative Neanderthal variant" of TKTL1 is present in modern human backgrounds. We dispute their argument that this genetic variant is responsible for brain differences in modern humans as opposed to Neanderthals.

MicroRNA-eQTLs in the developing human neocortex link miR-4707-3p expression to brain size.

Expression quantitative trait loci (eQTL) data have proven important for linking non-coding loci to protein-coding genes. But eQTL studies rarely measure microRNAs (miRNAs), small non-coding RNAs known to play a role in human brain development and neurogenesis. Here, we performed small-RNA sequencing across 212 mid-gestation human neocortical tissue samples, measured 907 expressed miRNAs, discovering 111 of which were novel, and identified 85 local-miRNA-eQTLs. Colocalization of miRNA-eQTLs with GWAS summary statistics yielded one robust colocalization of miR-4707-3p expression with educational attainment and brain size phenotypes, where the miRNA expression increasing allele was associated with decreased brain size. Exogenous expression of miR-4707-3p in primary human neural progenitor cells decreased expression of predicted targets and increased cell proliferation, indicating miR-4707-3p modulates progenitor gene regulation and cell fate decisions. Integrating miRNA-eQTLs with existing GWAS yielded evidence of a miRNA that may influence human brain size and function via modulation of neocortical brain development.

Somatic mosaicism reveals clonal distributions of neocortical development.

The structure of the human neocortex underlies species-specific traits and reflects intricate developmental programs. Here we sought to reconstruct processes that occur during early development by sampling adult human tissues. We analysed neocortical clones in a post-mortem human brain through a comprehensive assessment of brain somatic mosaicism, acting as neutral lineage recorders1,2. We combined the sampling of 25 distinct anatomic locations with deep whole-genome sequencing in a neurotypical deceased individual and confirmed results with 5 samples collected from each of three additional donors. We identified 259 bona fide mosaic variants from the index case, then deconvolved distinct geographical, cell-type and clade organizations across the brain and other organs. We found that clones derived after the accumulation of 90-200 progenitors in the cerebral cortex tended to respect the midline axis, well before the anterior-posterior or ventral-dorsal axes, representing a secondary hierarchy following the overall patterning of forebrain and hindbrain domains. Clones across neocortically derived cells were consistent with a dual origin from both dorsal and ventral cellular populations, similar to rodents, whereas the microglia lineage appeared distinct from other resident brain cells. Our data provide a comprehensive analysis of brain somatic mosaicism across the neocortex and demonstrate cellular origins and progenitor distribution patterns within the human brain.

The people behind the paper - Peter Kozulin, Rodrigo Suárez, Qiong-Yi Zhao, Linda Richards and Laura Fenlon.

The neocortex is unique to mammals and so, for evolutionary studies, researchers have compared eutherians and marsupials. A new paper in Development uncovers key differences in the timing of gene expression changes in the cortical development of the mouse and the similarly sized marsupial, the fat-tailed dunnart. We caught up with the authors from The University of Queensland, Australia, to find out more about their research and their future plans.

Temperature sensitivity of Notch signaling underlies species-specific developmental plasticity and robustness in amniote brains.

Ambient temperature significantly affects developmental timing in animals. The temperature sensitivity of embryogenesis is generally believed to be a consequence of the thermal dependency of cellular metabolism. However, the adaptive molecular mechanisms that respond to variations in temperature remain unclear. Here, we report species-specific thermal sensitivity of Notch signaling in the developing amniote brain. Transient hypothermic conditions increase canonical Notch activity and reduce neurogenesis in chick neural progenitors. Increased biosynthesis of phosphatidylethanolamine, a major glycerophospholipid components of the plasma membrane, mediates hypothermia-induced Notch activation. Furthermore, the species-specific thermal dependency of Notch signaling is associated with developmental robustness to altered Notch signaling. Our results reveal unique regulatory mechanisms for temperature-dependent neurogenic potentials that underlie developmental and evolutionary adaptations to a range of ambient temperatures in amniotes.

Divergent evolution of developmental timing in the neocortex revealed by marsupial and eutherian transcriptomes.

Only mammals evolved a neocortex, which integrates sensory-motor and cognitive functions. Significant diversifications in the cellular composition and connectivity of the neocortex occurred between the two main therian groups: marsupials and eutherians. However, the developmental mechanisms underlying these diversifications are largely unknown. Here, we compared the neocortical transcriptomes of Sminthopsis crassicaudata, a mouse-sized marsupial, with those of eutherian mice at two developmentally equivalent time points corresponding to deeper and upper layer neuron generation. Enrichment analyses revealed more mature gene networks in marsupials at the early stage, which reverted at the later stage, suggesting a more precocious but protracted neuronal maturation program relative to birth timing of cortical layers. We ranked genes expressed in different species and identified important differences in gene expression rankings between species. For example, genes known to be enriched in upper-layer cortical projection neuron subtypes, such as Cux1, Lhx2 and Satb2, likely relate to corpus callosum emergence in eutherians. These results show molecular heterochronies of neocortical development in Theria, and highlight changes in gene expression and cell type composition that may underlie neocortical evolution and diversification. This article has an associated 'The people behind the papers' interview.

Human neocortical expansion involves glutamatergic neuron diversification.

The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer's disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.

Dependence of Some Indicators of Brain Development in 30-Day-Old Rats on the Dynamics of Body Weight Gain in the Neonatal and Suckling Periods in Litters of Different Sizes.

We analyzed the dependence of the weight of the brain, its hemispheres, and morphometric parameters of the parietal cortex and the hippocampus in 30-day-old Wistar rats on their body weight at the age of 1, 7, 14, 21, and 30 days. All the animals were from medium-sized litters. In 6 litters (experiment), 6 rat pups were left in each litter 1 day after delivery; in 6 other litters (control), their number remained unchanged (8-13 pups). In both groups, a positive correlation was revealed between the brain weight and body weight at the age of 1, 7, 14, 21, and 30 days. At the same time, the body weight in rats aged 7, 14, 21, and 30 days and the brain and hemisphere weight at the age of 30 days in the experimental group was significantly greater than in the control group. Rats of the experimental group had higher numerical density of gliocytes in layer II and V of the neocortex and a greater ratio of glia/neurons in these layers. The cortical neurons in the experimental rats were larger than in the control in field I of the hippocampus (p<0.05) and in layer II (p<0.05) and layer V (p>0.05) of the neocortex. The neuronal nuclei in rats from reduced litters were significantly larger than in control animals.

Transient expansion of the expression region of Hsd11b1, encoding 11ß-hydroxysteroid dehydrogenase type 1, in the developing mouse neocortex.

Corticosteroids are stress-related hormones that maintain homeostasis. The most effective corticosteroids are corticosterone (CORT) in rodents and cortisol in primates. 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1; EC 1.1.1.146), encoded by Hsd11b1, is a key regulator of the local concentration of CORT/cortisol. Hsd11b1 expression in layer 5 of the primary somatosensory cortex has been shown in adult mice. However, its localization in the entire neocortex, especially during development, has not been fully addressed. Here, we established robust and dynamic expression profiles of Hsd11b1 in the developing mouse neocortex. Hsd11b1 was found mostly in pyramidal neurons. By retrograde tracing, we observed that some Hsd11b1-positive cells were projection neurons, indicating that at least some were excitatory. At postnatal day 0 (P0), Hsd11b1 was expressed in the deep layer of the somatosensory cortex. Then, from P3 to P8, the expression area expanded broadly; it was observed in layers 4 and 5, spanning the whole neocortex, including the primary motor cortex (M1) and the primary visual cortex (V1). The positive region gradually narrowed from P14 onwards and was ultimately limited to layer 5 of the somatosensory cortex at P26 and later. Furthermore, we administered CORT to nursing dams to increase the systemic CORT level of their pups. Here, we observed a reduced number of Hsd11b1-positive cells in the neocortex of these pups. Our observation suggests that Hsd11b1 expression in the developing neocortex is affected by systemic CORT levels. It is possible that stress on mothers influences the neocortical development of their children.

Inheritance and flexibility of cell polarity: a clue for understanding human brain development and evolution.

Cell polarity is fundamentally important for understanding brain development. Here, we hypothesize that the inheritance and flexibility of cell polarity during neocortex development could be implicated in neocortical evolutionary expansion. Molecular and morphological features of cell polarity may be inherited from one type of progenitor cell to the other and finally transmitted to neurons. Furthermore, key cell types, such as basal progenitors and neurons, exhibit a highly flexible polarity. We suggest that both inheritance and flexibility of cell polarity are implicated in the amplification of basal progenitors and tangential dispersion of neurons, which are key features of the evolutionary expansion of the neocortex.

Extensive Structural Remodeling of the Axonal Arbors of Parvalbumin Basket Cells during Development in Mouse Neocortex.

Parvalbumin-containing (PV+) basket cells are specialized cortical interneurons that regulate the activity of local neuronal circuits with high temporal precision and reliability. To understand how the PV+ interneuron connectivity underlying these functional properties is established during development, we used array tomography to map pairs of synaptically connected PV+ interneurons and postsynaptic neurons from the neocortex of mice of both sexes. We focused on the axon-myelin unit of the PV+ interneuron and quantified the number of synapses onto the postsynaptic neuron, length of connecting axonal paths, and their myelination at different time points between 2 weeks and 7 months of age. We find that myelination of the proximal axon occurs very rapidly during the third and, to a lesser extent, fourth postnatal weeks. The number of synaptic contacts made by the PV+ interneuron on its postsynaptic partner meanwhile is significantly reduced to about one-third by the end of the first postnatal month. The number of autapses, the synapses that PV+ interneurons form on themselves, however, remains constant throughout the examined period. Axon reorganizations continue beyond postnatal month 2, with the postsynaptic targets of PV+ interneurons gradually shifting to more proximal locations, and the length of axonal paths and their myelin becoming conspicuously uniform per connection. These continued microcircuit refinements likely provide the structural substrate for the robust inhibitory effects and fine temporal precision of adult PV+ basket cells.SIGNIFICANCE STATEMENT The axon of adult parvalbumin-containing (PV+) interneurons is highly specialized for fast and reliable neurotransmission. It is myelinated and forms synapses mostly onto the cell bodies and proximal dendrites of postsynaptic neurons for maximal impact. In this study, we follow the development of the PV+ interneuron axon, its myelination and synapse formation, revealing a rapid sequence of axonal reorganization, myelination of the PV+ interneuron proximal axon, and pruning of almost two-thirds of the synapses in an individual connection. This is followed by a prolonged period of axon refinement and additional myelination leading to a remarkable precision of connections in the adult mouse cortex, consistent with the temporal precision and fidelity of PV+ interneuron action.

ZBTB20 is crucial for the specification of a subset of callosal projection neurons and astrocytes in the mammalian neocortex.

Neocortical progenitor cells generate subtypes of excitatory projection neurons in sequential order followed by the generation of astrocytes. The transcription factor zinc finger and BTB domain-containing protein 20 (ZBTB20) has been implicated in regulation of cell specification during neocortical development. Here, we show that ZBTB20 instructs the generation of a subset of callosal projections neurons in cortical layers II/III in mouse. Conditional deletion of Zbtb20 in cortical progenitors, and to a lesser degree in differentiating neurons, leads to an increase in the number of layer IV neurons at the expense of layer II/III neurons. Astrogliogenesis is also affected in the mutants with an increase in the number of a specific subset of astrocytes expressing GFAP. Astrogliogenesis is more severely disrupted by a ZBTB20 protein containing dominant mutations linked to Primrose syndrome, suggesting that ZBTB20 acts in concert with other ZBTB proteins that were also affected by the dominant-negative protein to instruct astrogliogenesis. Overall, our data suggest that ZBTB20 acts both in progenitors and in postmitotic cells to regulate cell fate specification in the mammalian neocortex.

How neural stem cells contribute to neocortex development.

The mammalian neocortex is the seat of higher cognitive functions, such as thinking and language in human. A hallmark of the neocortex are the cortical neurons, which are generated from divisions of neural progenitor cells (NPCs) during development, and which constitute a key feature of the well-organized layered structure of the neocortex. Proper formation of neocortex structure requires an orchestrated cellular behavior of different cortical NPCs during development, especially during the process of cortical neurogenesis. Here, we review the great diversity of NPCs and their contribution to the development of the neocortex. First, we review the categorization of NPCs into different classes and types based on their cell biological features, and discuss recent advances in characterizing marker expression and cell polarity features in the different types of NPCs. Second, we review the different modes of cell divisions that NPCs undergo and discuss the importance of the balance between proliferation and differentiation of NPCs in neocortical development. Third, we review the different proliferative capacities among different NPC types and among the same type of NPC in different mammalian species. Dissecting the differences between NPC types and differences among mammalian species is beneficial to further understand the development and the evolutionary expansion of the neocortex and may open up new therapeutic avenues for neurodevelopmental and psychiatric disorders.

Reelin restricts dendritic growth of interneurons in the neocortex.

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation.

Brain-trait-associated variants impact cell-type-specific gene regulation during neurogenesis.

Interpretation of the function of non-coding risk loci for neuropsychiatric disorders and brain-relevant traits via gene expression and alternative splicing quantitative trait locus (e/sQTL) analyses is generally performed in bulk post-mortem adult tissue. However, genetic risk loci are enriched in regulatory elements active during neocortical differentiation, and regulatory effects of risk variants may be masked by heterogeneity in bulk tissue. Here, we map e/sQTLs, and allele-specific expression in cultured cells representing two major developmental stages, primary human neural progenitors (n = 85) and their sorted neuronal progeny (n = 74), identifying numerous loci not detected in either bulk developing cortical wall or adult cortex. Using colocalization and genetic imputation via transcriptome-wide association, we uncover cell-type-specific regulatory mechanisms underlying risk for brain-relevant traits that are active during neocortical differentiation. Specifically, we identified a progenitor-specific eQTL for CENPW co-localized with common variant associations for cortical surface area and educational attainment.

Differential Expression Levels of Sox9 in Early Neocortical Radial Glial Cells Regulate the Decision between Stem Cell Maintenance and Differentiation.

Radial glial progenitor cells (RGCs) in the dorsal telencephalon directly or indirectly produce excitatory projection neurons and macroglia of the neocortex. Recent evidence shows that the pool of RGCs is more heterogeneous than originally thought and that progenitor subpopulations can generate particular neuronal cell types. Using single-cell RNA sequencing, we have studied gene expression patterns of RGCs with different neurogenic behavior at early stages of cortical development. At this early age, some RGCs rapidly produce postmitotic neurons, whereas others self-renew and undergo neurogenic divisions at a later age. We have identified candidate genes that are differentially expressed among these early RGC subpopulations, including the transcription factor Sox9. Using in utero electroporation in embryonic mice of either sex, we demonstrate that elevated Sox9 expression in progenitors affects RGC cell cycle duration and leads to the generation of upper layer cortical neurons. Our data thus reveal molecular differences between progenitor cells with different neurogenic behavior at early stages of corticogenesis and indicates that Sox9 is critical for the maintenance of RGCs to regulate the generation of upper layer neurons.SIGNIFICANCE STATEMENT The existence of heterogeneity in the pool of RGCs and its relationship with the generation of cellular diversity in the cerebral cortex has been an interesting topic of debate for many years. Here we describe the existence of RGCs with reduced neurogenic behavior at early embryonic ages presenting a particular molecular signature. This molecular signature consists of differential expression of some genes including the transcription factor Sox9, which has been found to be a specific regulator of this subpopulation of progenitor cells. Functional experiments perturbing expression levels of Sox9 reveal its instructive role in the regulation of the neurogenic behavior of RGCs and its relationship with the generation of upper layer projection neurons at later ages.

Biphasic Roles of Hedgehog Signaling in the Production and Self-Renewal of Outer Radial Glia in the Ferret Cerebral Cortex.

The neocortex, the center for higher brain function, emerged in mammals and expanded in the course of evolution. The expansion of outer radial glia (oRGs) and intermediate progenitor cells (IPCs) plays key roles in the expansion and consequential folding of the neocortex. Therefore, understanding the mechanisms of oRG and IPC expansion is important for understanding neocortical development and evolution. By using mice and human cerebral organoids, we previously revealed that hedgehog (HH) signaling expands oRGs and IPCs. Nevertheless, it remained to be determined whether HH signaling expanded oRGs and IPCs in vivo in gyrencephalic species, in which oRGs and IPCs are naturally expanded. Here, we show that HH signaling is necessary and sufficient to expand oRGs and IPCs in ferrets, a gyrencephalic species, through conserved cellular mechanisms. HH signaling increases oRG-producing division modes of ventricular radial glia (vRGs), oRG self-renewal, and IPC proliferation. Notably, HH signaling affects vRG division modes only in an early restricted phase before superficial-layer neuron production peaks. Beyond this restricted phase, HH signaling promotes oRG self-renewal. Thus, HH signaling expands oRGs and IPCs in two distinct but continuous phases during cortical development.

Zbed3 Is Indispensable for Wnt Signaling Regulation of Cortical Layers Formation in Developing Brain.

Wnt/ß-catenin signaling plays multiple important roles during mammalian brain development, and it regulates the proliferation and differentiation of neural progenitors in a context-dependent manner and affects neocortex layer formation. However, the specific role of Wnt/ß-catenin in neuronal layer fate determination in the neocortex is still unclear. Here, we report that Zbed3, which is a positive regulator of Wnt/ß-catenin signaling, colocalizes with ß-catenin at the endfeet of radial glia in the ventricular zone of embryo mouse neocortex. Overexpression and knockdown of Zbed3 increased and decreased the activity of Wnt/ß-catenin signaling in the neocortex, respectively. Interestingly, knockdown of Zbed3 in vivo could significantly shift neuronal fates from deep layers to upper layers but is not required for the proliferation and differentiation of neural progenitors. Overexpression of Zbed3 led to increased generation of deep-layer neurons without impairing cell cycle exit of neural progenitors. More importantly, knockdown of Zbed3 could effectively block the effects of the ectopic expression of stabilized ß-catenin on neocortex layer formation. Hence, our results demonstrate that Zbed3 is indispensable for Wnt/ß-catenin signaling regulating neuronal layer fates in the developing brain.