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The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response.

The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.

Nuclear compartments are thought to play a role in three-dimensional genome organization and gene expression. In mammalian brain, the architecture and dynamics of nuclear compartment-associated genome organization is not known. In this study, we developed Genome Organization using CUT and RUN Technology (GO-CaRT) to map genomic interactions with two nuclear compartments-the nuclear lamina and nuclear speckles-from different regions of the developing mouse, macaque and human brain. Lamina-associated domain (LAD) architecture in cells in vivo is distinct from that of cultured cells, including major differences in LADs previously considered to be cell type invariant. In the mouse and human forebrain, dorsal and ventral neural precursor cells have differences in LAD architecture that correspond to their regional identity. LADs in the human and mouse cortex contain transcriptionally highly active sub-domains characterized by broad depletion of histone-3-lysine-9 dimethylation. Evolutionarily conserved LADs in human, macaque and mouse brain are enriched for transcriptionally active neural genes associated with synapse function. By integrating GO-CaRT maps with genome-wide association study data, we found speckle-associated domains to be enriched for schizophrenia risk loci, indicating a physical relationship between these disease-associated genetic variants and a specific nuclear structure. Our work provides a framework for understanding the relationship between distinct nuclear compartments and genome function in brain development and disease.

The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.

Aberrant aggregation of the RNA-binding protein TDP-43 in neurons is a hallmark of frontotemporal lobar degeneration caused by haploinsufficiency in the gene encoding progranulin1,2. However, the mechanism leading to TDP-43 proteinopathy remains unclear. Here we use single-nucleus RNA sequencing to show that progranulin deficiency promotes microglial transition from a homeostatic to a disease-specific state that causes endolysosomal dysfunction and neurodegeneration in mice. These defects persist even when Grn-/- microglia are cultured ex vivo. In addition, single-nucleus RNA sequencing reveals selective loss of excitatory neurons at disease end-stage, which is characterized by prominent nuclear and cytoplasmic TDP-43 granules and nuclear pore defects. Remarkably, conditioned media from Grn-/- microglia are sufficient to promote TDP-43 granule formation, nuclear pore defects and cell death in excitatory neurons via the complement activation pathway. Consistent with these results, deletion of the genes encoding C1qa and C3 mitigates microglial toxicity and rescues TDP-43 proteinopathy and neurodegeneration. These results uncover previously unappreciated contributions of chronic microglial toxicity to TDP-43 proteinopathy during neurodegeneration.

BackgroundStudies of individuals with autism spectrum disorder (ASD) have revealed a strong multigenic basis with the identification of hundreds of ASD susceptibility genes. ASD is characterized by social deficits and a range of other phenotypes, implicating complex genetics and involvement of a variety of brain regions. However, how mutations and mis-expression of select gene sets are associated with the behavioral components of ASD remains unknown. We reasoned that for genes to be associated with ASD core behaviors they must be: (1) expressed in brain regions relevant to ASD social behaviors and (2) expressed during the ASD susceptible window of brain development.MethodsFocusing on the amygdala, a brain region whose dysfunction has been highly implicated in the social component of ASD, we mined publicly available gene expression databases to identify ASD-susceptibility genes expressed during human and mouse amygdala development. We found that a large cohort of known ASD susceptibility genes is expressed in the developing human and mouse amygdala. We further performed analysis of single-nucleus RNA-seq (snRNA-seq) data from microdissected amygdala tissue from five ASD and five control human postmortem brains ranging in age from 4 to 20 years to elucidate cell type specificity of amygdala-expressed genes and their dysregulation in ASD.ResultsOur analyses revealed that of the high-ranking ASD susceptibility genes, 80 are expressed in both human and mouse amygdala during fetal to early postnatal stages of development. Our human snRNA-seq analyses revealed cohorts of genes with altered expression in the ASD amygdala postnatally, especially within excitatory neurons, with dysregulated expression of seven genes predicted from our datamining pipeline.LimitationsWe were limited by the ages for which we were able to obtain human tissue; therefore, the results from our datamining pipeline approach will require validation, to the extent possible, in human tissue from earlier developmental stages.ConclusionsOur pipeline narrows down the number of amygdala-expressed genes possibly involved in the social pathophysiology of ASD. Our human single-nucleus gene expression analyses revealed that ASD is characterized by changes in gene expression in specific cell types in the early postnatal amygdala.

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.

To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.

Neural stem cells (NSCs) in the prenatal neocortex progressively generate different subtypes of glutamatergic projection neurons. Following that, NSCs have a major switch in their progenitor properties and produce γ-aminobutyric acid (GABAergic) interneurons for the olfactory bulb (OB), cortical oligodendrocytes, and astrocytes. Herein, we provide evidence for the molecular mechanism that underlies this switch in the state of neocortical NSCs. We show that, at around E16.5, mouse neocortical NSCs start to generate GSX2-expressing (GSX2+) intermediate progenitor cells (IPCs). In vivo lineage-tracing study revealed that GSX2+ IPC population gives rise not only to OB interneurons but also to cortical oligodendrocytes and astrocytes, suggesting that they are a tri-potential population. We demonstrated that Sonic hedgehog signaling is both necessary and sufficient for the generation of GSX2+ IPCs by reducing GLI3R protein levels. Using single-cell RNA sequencing, we identify the transcriptional profile of GSX2+ IPCs and the process of the lineage switch of cortical NSCs.

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.

Multiple sclerosis (MS) is a neuroinflammatory disease with a relapsing-remitting disease course at early stages, distinct lesion characteristics in cortical grey versus subcortical white matter and neurodegeneration at chronic stages. Here we used single-nucleus RNA sequencing to assess changes in expression in multiple cell lineages in MS lesions and validated the results using multiplex in situ hybridization. We found selective vulnerability and loss of excitatory CUX2-expressing projection neurons in upper-cortical layers underlying meningeal inflammation; such MS neuron populations exhibited upregulation of stress pathway genes and long non-coding RNAs. Signatures of stressed oligodendrocytes, reactive astrocytes and activated microglia mapped most strongly to the rim of MS plaques. Notably, single-nucleus RNA sequencing identified phagocytosing microglia and/or macrophages by their ingestion and perinuclear import of myelin transcripts, confirmed by functional mouse and human culture assays. Our findings indicate lineage- and region-specific transcriptomic changes associated with selective cortical neuron damage and glial activation contributing to progression of MS lesions.

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversity not captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, but not mouse, neocortex, and inhibiting HTR2A receptors in human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmitter signaling during neurogenesis in the developing human neocortex and highlight evolutionarily divergent mechanisms of physiological signaling.

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

BackgroundHigh throughput methods for profiling the transcriptomes of single cells have recently emerged as transformative approaches for large-scale population surveys of cellular diversity in heterogeneous primary tissues. However, the efficient generation of such atlases will depend on sufficient sampling of diverse cell types while remaining cost-effective to enable a comprehensive examination of organs, developmental stages, and individuals.ResultsTo examine the relationship between sampled cell numbers and transcriptional heterogeneity in the context of unbiased cell type classification, we explored the population structure of a publicly available 1.3 million cell dataset from E18.5 mouse brain and validated our findings in published data from adult mice. We propose a computational framework for inferring the saturation point of cluster discovery in a single-cell mRNA-seq experiment, centered around cluster preservation in downsampled datasets. In addition, we introduce a "complexity index," which characterizes the heterogeneity of cells in a given dataset. Using Cajal-Retzius cells as an example of a limited complexity dataset, we explored whether the detected biological distinctions relate to technical clustering. Surprisingly, we found that clustering distinctions carrying biologically interpretable meaning are achieved with far fewer cells than the originally sampled, though technical saturation of rare populations such as Cajal-Retzius cells is not achieved. We additionally validated these findings with a recently published atlas of cell types across mouse organs and again find using subsampling that a much smaller number of cells recapitulates the cluster distinctions of the complete dataset.ConclusionsTogether, these findings suggest that most of the biologically interpretable cell types from the 1.3 million cell database can be recapitulated by analyzing 50,000 randomly selected cells, indicating that instead of profiling few individuals at high "cellular coverage," cell atlas studies may instead benefit from profiling more individuals, or many time points at lower cellular coverage and then further enriching for populations of interest. This strategy is ideal for scenarios where cost and time are limited, though extremely rare populations of interest (

The neocortex of primates, including humans, contains more abundant and diverse inhibitory neurons compared with rodents, but the molecular foundations of these observations are unknown. Through integrative gene coexpression analysis, we determined a consensus transcriptional profile of GABAergic neurons in mid-gestation human neocortex. By comparing this profile to genes expressed in GABAergic neurons purified from neonatal mouse neocortex, we identified conserved and distinct aspects of gene expression in these cells between the species. We show here that the calcium-binding protein secretagogin (SCGN) is robustly expressed by neocortical GABAergic neurons derived from caudal ganglionic eminences (CGE) and lateral ganglionic eminences during human but not mouse brain development. Through electrophysiological and morphometric analyses, we examined the effects of SCGN expression on GABAergic neuron function and form. Forced expression of SCGN in CGE-derived mouse GABAergic neurons significantly increased total neurite length and arbor complexity following transplantation into mouse neocortex, revealing a molecular pathway that contributes to morphological differences in these cells between rodents and primates.

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

BACKGROUND:Tumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue. RESULTS:We perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells. CONCLUSIONS:We conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.

A comprehensive characterization of neuronal cell types, their distributions, and patterns of connectivity is critical for understanding the properties of neural circuits and how they generate behaviors. Here we review the experiences of the BRAIN Initiative Cell Census Consortium, ten pilot projects funded by the U.S. BRAIN Initiative, in developing, validating, and scaling up emerging genomic and anatomical mapping technologies for creating a complete inventory of neuronal cell types and their connections in multiple species and during development. These projects lay the foundation for a larger and longer-term effort to generate whole-brain cell atlases in species including mice and humans.

Understanding the development and dysfunction of the human brain is a major goal of neurobiology. Much of our current understanding of human brain development has been derived from the examination of post-mortem and pathological specimens, bolstered by observations of developing non-human primates and experimental studies focused largely on mouse models. However, these tissue specimens and model systems cannot fully capture the unique and dynamic features of human brain development. Recent advances in stem cell technologies that enable the generation of human brain organoids from pluripotent stem cells (PSCs) promise to profoundly change our understanding of the development of the human brain and enable a detailed study of the pathogenesis of inherited and acquired brain diseases.

Long non-coding RNAs (lncRNAs) are a diverse and poorly conserved category of transcripts that have expanded greatly in primates, particularly in the brain. We identified an lncRNA, which has acquired 16 microRNA response elements for miR-143-3p in the Catarrhini branch of primates. This lncRNA, termed LncND (neurodevelopment), is expressed in neural progenitor cells and then declines in neurons. Binding and release of miR-143-3p by LncND control the expression of Notch receptors. LncND expression is enriched in radial glia cells (RGCs) in the ventricular and subventricular zones of developing human brain. Downregulation in neuroblastoma cells reduced cell proliferation and induced neuronal differentiation, an effect phenocopied by miR-143-3p overexpression. Gain of function of LncND in developing mouse cortex led to an expansion of PAX6+ RGCs. These findings support a role for LncND in miRNA-mediated regulation of Notch signaling within the neural progenitor pool in primates that may have contributed to the expansion of cerebral cortex.

The recent outbreak of Zika virus (ZIKV) in Brazil has been linked to substantial increases in fetal abnormalities and microcephaly. However, information about the underlying molecular and cellular mechanisms connecting viral infection to these defects remains limited. In this study we have examined the expression of receptors implicated in cell entry of several enveloped viruses including ZIKV across diverse cell types in the developing brain. Using single-cell RNA-seq and immunohistochemistry, we found that the candidate viral entry receptor AXL is highly expressed by human radial glial cells, astrocytes, endothelial cells, and microglia in developing human cortex and by progenitor cells in developing retina. We also show that AXL expression in radial glia is conserved in developing mouse and ferret cortex and in human stem cell-derived cerebral organoids, highlighting multiple experimental systems that could be applied to study mechanisms of ZIKV infectivity and effects on brain development.

γ-Aminobutyric acid (GABA) is the major inhibitory transmitter in the mature brain but is excitatory in the developing cortex. We found that mouse zona incerta (ZI) projection neurons form a GABAergic axon plexus in neonatal cortical layer 1, making synapses with neurons in both deep and superficial layers. A similar depolarizing GABAergic plexus exists in the developing human cortex. Selectively silencing mouse ZI GABAergic neurons at birth decreased synaptic activity and apical dendritic complexity of cortical neurons. The ZI GABAergic projection becomes inhibitory with maturation and can block epileptiform activity in the adult brain. These data reveal an early-developing GABAergic projection from the ZI to cortical layer 1 that is essential for proper development of cortical neurons and balances excitation with inhibition in the adult cortex.

During mammalian neocortical development, newborn excitatory and inhibitory neurons must migrate over long distances to reach their final positions within the cortical plate. In the lissencephalic rodent brain, pyramidal neurons are born in the ventricular and subventricular zones of the pallium and migrate along radial glia fibers to reach the appropriate cortical layer. Although much less is known about neuronal migration in species with a gyrencephalic cortex, retroviral studies in the ferret and primate suggest that, unlike the rodent, pyramidal neurons do not follow strict radial pathways and instead can disperse horizontally. However, the means by which pyramidal neurons laterally disperse remain unknown. In this study, we identified a viral labeling technique for visualizing neuronal migration in the ferret, a gyrencephalic carnivore, and found that migration was predominantly radial at early postnatal ages. In contrast, neurons displayed more tortuous migration routes with a decreased frequency of cortical plate-directed migration at later stages of neurogenesis concomitant with the start of brain folding. This was accompanied by neurons migrating sequentially along several different radial glial fibers, suggesting a mode by which pyramidal neurons may laterally disperse in a folded cortex. These findings provide insight into the migratory behavior of neurons in gyrencephalic species and provide a framework for using nonrodent model systems for studying neuronal migration disorders.Significance statementElucidating neuronal migration dynamics in the gyrencephalic, or folded, cortex is important for understanding neurodevelopmental disorders. Similar to the rodent, we found that neuronal migration was predominantly radial at early postnatal ages in the gyrencephalic ferret cortex. Interestingly, ferret neurons displayed more tortuous migration routes and a decreased frequency of radial migration at later ages coincident with the start of cortical folding. We found that ferret neurons use several different radial glial fibers as migratory guides, including those belonging to the recently described outer radial glia, suggesting a mechanism by which ferret neurons disperse laterally. It is likely that excitatory neurons horizontally disperse in other gyrencephalic mammals, including the primate, suggesting an important modification to the current model deduced primarily from the rodent.

The mammalian neocortex is composed of two major neuronal cell types with distinct origins: excitatory pyramidal neurons and inhibitory interneurons, generated in dorsal and ventral progenitor zones of the embryonic telencephalon, respectively. Thus, inhibitory neurons migrate relatively long distances to reach their destination in the developing forebrain. The role of lineage in the organization and circuitry of interneurons is still not well understood. Utilizing a combination of genetics, retroviral fate mapping, and lineage-specific retroviral barcode labeling, we find that clonally related interneurons can be widely dispersed while unrelated interneurons can be closely clustered. These data suggest that migratory mechanisms related to the clustering of interneurons occur largely independent of their clonal origin.

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.

While thousands of long noncoding RNAs (lncRNAs) have been identified, few lncRNAs that control neural stem cell (NSC) behavior are known. Here, we identify Pinky (Pnky) as a neural-specific lncRNA that regulates neurogenesis from NSCs in the embryonic and postnatal brain. In postnatal NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Pnky interacts with the splicing regulator PTBP1, and PTBP1 knockdown also enhances neurogenesis. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. These data thus unveil Pnky as a conserved lncRNA that interacts with a key RNA processing factor and regulates neurogenesis from embryonic and postnatal NSC populations.

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that the Ink4a/Arf (also known as Cdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passage in vitro, wild-type postnatal cortical astroglia become progressively resistant to Dlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence of Ink4a/Arf-deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced from Ink4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16(Ink4a) and p19(Arf) indicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient for Ink4a/Arf also exhibit greatly enhanced transcription factor-induced neuronal induction. These data indicate that Ink4a/Arf is a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes.

Many neurologic and psychiatric disorders are marked by imbalances between neural excitation and inhibition. In the cerebral cortex, inhibition is mediated largely by GABAergic (γ-aminobutyric acid-secreting) interneurons, a cell type that originates in the embryonic ventral telencephalon and populates the cortex through long-distance tangential migration. Remarkably, when transplanted from embryos or in vitro culture preparations, immature interneurons disperse and integrate into host brain circuits, both in the cerebral cortex and in other regions of the central nervous system. These features make interneuron transplantation a powerful tool for the study of neurodevelopmental processes such as cell specification, cell death, and cortical plasticity. Moreover, interneuron transplantation provides a novel strategy for modifying neural circuits in rodent models of epilepsy, Parkinson's disease, mood disorders, and chronic pain.

The dramatic increase in neocortical size and folding during mammalian brain evolution has been attributed to the elaboration of the subventricular zone (SVZ) and the associated increase in neural progenitors. However, recent studies have shown that SVZ size and the abundance of resident progenitors do not directly predict cortical topography, suggesting that complex behaviors of the progenitors themselves may contribute to the overall size and shape of the adult cortex. Using time-lapse imaging, we examined the dynamic behaviors of SVZ progenitors in the ferret, a gyrencephalic carnivore, focusing our analysis on outer radial glial cells (oRGs). We identified a substantial population of oRGs by marker expression and their unique mode of division, termed mitotic somal translocation (MST). Ferret oRGs exhibited diverse behaviors in terms of division location, cleavage angle, and MST distance, as well as fiber orientation and dynamics. We then examined the human fetal cortex and found that a subset of human oRGs displayed similar characteristics, suggesting that diversity in oRG behavior may be a general feature. Similar to the human, ferret oRGs underwent multiple rounds of self-renewing divisions but were more likely to undergo symmetric divisions that expanded the oRG population, as opposed to producing intermediate progenitor cells (IPCs). Differences in oRG behaviors, including proliferative potential and daughter cell fates, may contribute to variations in cortical structure between mammalian species.

A three-dimensional culture of cortical tissues derived from pluripotent stem cells offers an opportunity to model human brain development and disorders. In a recent issue of Nature, Lancaster et al. describe a new method for generating cerebral organoids in a dish and use it to model microcephaly.

Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. However, most differentiated populations exhibit immature properties of an early embryonic stage, raising concerns about their ability to model and treat disease. Here, we report the directed differentiation of hPSCs into medial ganglionic eminence (MGE)-like progenitors and their maturation into forebrain type interneurons. We find that early-stage progenitors progress via a radial glial-like stem cell enriched in the human fetal brain. Both in vitro and posttransplantation into the rodent cortex, the MGE-like cells develop into GABAergic interneuron subtypes with mature physiological properties along a prolonged intrinsic timeline of up to 7 months, mimicking endogenous human neural development. MGE-derived cortical interneuron deficiencies are implicated in a broad range of neurodevelopmental and degenerative disorders, highlighting the importance of these results for modeling human neural development and disease.

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

Polarization of mammalian neurons with a specified axon requires precise regulation of microtubule and actin dynamics in the developing neurites. Here we show that mammalian partition defective 3 (mPar3), a key component of the Par polarity complex that regulates the polarization of many cell types including neurons, directly regulates microtubule stability and organization. The N-terminal portion of mPar3 exhibits strong microtubule binding, bundling, and stabilization activity, which can be suppressed by its C-terminal portion via an intramolecular interaction. Interestingly, the intermolecular oligomerization of mPar3 is able to relieve the intramolecular interaction and thereby promote microtubule bundling and stabilization. Furthermore, disruption of this microtubule regulatory activity of mPar3 impairs its function in axon specification. Together, these results demonstrate a role for mPar3 in directly regulating microtubule organization that is crucial for neuronal polarization.

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+) cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+) cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while enhancer 1056 is active in Olig2(+) cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

Radial glial progenitor cells exhibit bidirectional cell cycle-dependent nuclear oscillations. The purpose and underlying mechanism of this unusual 'interkinetic nuclear migration' are poorly understood. We investigated the basis for this behavior by live imaging of nuclei, centrosomes and microtubules in embryonic rat brain slices, coupled with the use of RNA interference (RNAi) and the myosin inhibitor blebbistatin. We found that nuclei migrated independent of centrosomes and unidirectionally away from or toward the ventricular surface along microtubules, which were uniformly oriented from the ventricular surface to the pial surface of the brain. RNAi directed against cytoplasmic dynein specifically inhibited nuclear movement toward the apical surface. An RNAi screen of kinesin genes identified Kif1a, a member of the kinesin-3 family, as the motor for basally directed nuclear movement. These observations provide direct evidence that kinesins are involved in nuclear migration and neurogenesis and suggest that a cell cycle-dependent switch between distinct microtubule motors drives interkinetic nuclear migration.

Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.

Summary: About 30% of patient with epilepsy do not respond to available antiepileptic drugs. In addition to seizure suppression, novel approaches are needed to prevent or alleviate epileptogenic process after various types of brain injuries. The use of cell transplants as factories to produce endogeneous anticonvulsants or as bricks to repair abnormal ictogenic and epileptogenic neuronal circuits has generated hope that cell-based therapies could become a novel therapeutic category in the treatment arsenal of epilepsy. Herein we summarize the current status and future perspectives of cell-based therapies in the treatment of epilepsy. [Copyright &y& Elsevier]