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Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Most genetic studies consider autism spectrum disorder (ASD) and developmental disorder (DD) separately despite overwhelming comorbidity and shared genetic etiology. Here, we analyzed de novo variants (DNVs) from 15,560 ASD (6,557 from SPARK) and 31,052 DD trios independently and also combined as broader neurodevelopmental disorders (NDDs) using three models. We identify 615 NDD candidate genes (false discovery rate [FDR] < 0.05) supported by ≥1 models, including 138 reaching Bonferroni exome-wide significance (P < 3.64e-7) in all models. The genes group into five functional networks associating with different brain developmental lineages based on single-cell nuclei transcriptomic data. We find no evidence for ASD-specific genes in contrast to 18 genes significantly enriched for DD. There are 53 genes that show mutational bias, including enrichments for missense (n = 41) or truncating (n = 12) DNVs. We also find 10 genes with evidence of male- or female-bias enrichment, including 4 X chromosome genes with significant female burden (DDX3X, MECP2, WDR45, and HDAC8). This large-scale integrative analysis identifies candidates and functional subsets of NDD genes.

The Internet of Things (IoT) provides a simple framework to control online devices easily. IoT is now a commonplace tool used by technology companies but is rarely used in biology experiments. IoT can benefit cloud biology research through alarm notifications, automation, and the real-time monitoring of experiments. We developed an IoT architecture to control biological devices and implemented it in lab experiments. Lab devices for electrophysiology, microscopy, and microfluidics were created from the ground up to be part of a unified IoT architecture. The system allows each device to be monitored and controlled from an online web tool. We present our IoT architecture so other labs can replicate it for their own experiments.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Progenitors of the developing human neocortex reside in the ventricular and outer subventricular zones (VZ and OSVZ, respectively). However, whether cells derived from these niches have similar developmental fates is unknown. By performing fate mapping in primary human tissue, we demonstrate that astrocytes derived from these niches populate anatomically distinct layers. Cortical plate astrocytes emerge from VZ progenitors and proliferate locally, while putative white matter astrocytes are morphologically heterogeneous and emerge from both VZ and OSVZ progenitors. Furthermore, via single-cell sequencing of morphologically defined astrocyte subtypes using Patch-seq, we identify molecular distinctions between VZ-derived cortical plate astrocytes and OSVZ-derived white matter astrocytes that persist into adulthood. Together, our study highlights a complex role for cell lineage in the diversification of human neocortical astrocytes.

Parvalbumin-positive (PV+) interneurons play a critical role in maintaining circuit rhythm in the brain, and their reduction is implicated in autism spectrum disorders. Animal studies demonstrate that maternal immune activation (MIA) leads to reduced PV+ interneurons in the somatosensory cortex and autism-like behaviors. However, the underlying molecular mechanisms remain largely unknown. Here, we show that MIA down-regulates microglial Gpr56 expression in fetal brains in an interleukin-17a-dependent manner and that conditional deletion of microglial Gpr56 [Gpr56 conditional knockout (cKO)] mimics MIA-induced PV+ interneuron defects and autism-like behaviors in offspring. We further demonstrate that elevated microglial tumor necrosis factor-α expression is the underlying mechanism by which MIA and Gpr56 cKO impair interneuron generation. Genetically restoring Gpr56 expression in microglia ameliorates PV+ interneuron deficits and autism-like behaviors in MIA offspring. Together, our study demonstrates that microglial GPR56 plays an important role in PV+ interneuron development and serves as a salient target of MIA-induced neurodevelopmental disorders.

Cerebrovascular diseases are a leading cause of death and neurologic disability. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of cerebrovascular cells in humans. We profiled transcriptomes of 181,388 cells to define a cell atlas of the adult human cerebrovasculature, including endothelial cell molecular signatures with arteriovenous segmentation and expanded perivascular cell diversity. By leveraging this reference, we investigated cellular and molecular perturbations in brain arteriovenous malformations, which are a leading cause of stroke in young people, and identified pathologic endothelial transformations with abnormal vascular patterning and the ontology of vascularly derived inflammation. We illustrate the interplay between vascular and immune cells that contributes to brain hemorrhage and catalog opportunities for targeting angiogenic and inflammatory programs in vascular malformations.

Neuroanatomists have long speculated that expanded primate brains contain an increased morphological diversity of inhibitory neurons (INs)1, and recent studies have identified primate-specific neuronal populations at the molecular level2. However, we know little about the developmental mechanisms that specify evolutionarily novel cell types in the brain. Here, we reconstruct gene expression trajectories specifying INs generated throughout the neurogenic period in macaques and mice by analysing the transcriptomes of 250,181 cells. We find that the initial classes of INs generated prenatally are largely conserved among mammals. Nonetheless, we identify two contrasting developmental mechanisms for specifying evolutionarily novel cell types during prenatal development. First, we show that recently identified primate-specific TAC3 striatal INs are specified by a unique transcriptional programme in progenitors followed by induction of a distinct suite of neuropeptides and neurotransmitter receptors in new-born neurons. Second, we find that multiple classes of transcriptionally conserved olfactory bulb (OB)-bound precursors are redirected to expanded primate white matter and striatum. These classes include a novel peristriatal class of striatum laureatum neurons that resemble dopaminergic periglomerular cells of the OB. We propose an evolutionary model in which conserved initial classes of neurons supplying the smaller primate OB are reused in the enlarged striatum and cortex. Together, our results provide a unified developmental taxonomy of initial classes of mammalian INs and reveal multiple developmental mechanisms for neural cell type evolution.

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.

The cerebral cortex is a cellularly complex structure comprising a rich diversity of neuronal and glial cell types. Cortical neurons can be broadly categorized into two classes-excitatory neurons that use the neurotransmitter glutamate, and inhibitory interneurons that use γ-aminobutyric acid (GABA). Previous developmental studies in rodents have led to a prevailing model in which excitatory neurons are born from progenitors located in the cortex, whereas cortical interneurons are born from a separate population of progenitors located outside the developing cortex in the ganglionic eminences1-5. However, the developmental potential of human cortical progenitors has not been thoroughly explored. Here we show that, in addition to excitatory neurons and glia, human cortical progenitors are also capable of producing GABAergic neurons with the transcriptional characteristics and morphologies of cortical interneurons. By developing a cellular barcoding tool called 'single-cell-RNA-sequencing-compatible tracer for identifying clonal relationships' (STICR), we were able to carry out clonal lineage tracing of 1,912 primary human cortical progenitors from six specimens, and to capture both the transcriptional identities and the clonal relationships of their progeny. A subpopulation of cortically born GABAergic neurons was transcriptionally similar to cortical interneurons born from the caudal ganglionic eminence, and these cells were frequently related to excitatory neurons and glia. Our results show that individual human cortical progenitors can generate both excitatory neurons and cortical interneurons, providing a new framework for understanding the origins of neuronal diversity in the human cortex.

During neurogenesis, mitotic progenitor cells lining the ventricles of the embryonic mouse brain undergo their final rounds of cell division, giving rise to a wide spectrum of postmitotic neurons and glia1,2. The link between developmental lineage and cell-type diversity remains an open question. Here we used massively parallel tagging of progenitors to track clonal relationships and transcriptomic signatures during mouse forebrain development. We quantified clonal divergence and convergence across all major cell classes postnatally, and found diverse types of GABAergic neuron that share a common lineage. Divergence of GABAergic clones occurred during embryogenesis upon cell-cycle exit, suggesting that differentiation into subtypes is initiated as a lineage-dependent process at the progenitor cell level.

SummaryAs the use of single-cell technologies has grown, so has the need for tools to explore these large, complicated datasets. The UCSC Cell Browser is a tool that allows scientists to visualize gene expression and metadata annotation distribution throughout a single-cell dataset or multiple datasets.Availability and implementationWe provide the UCSC Cell Browser as a free website where scientists can explore a growing collection of single-cell datasets and a freely available python package for scientists to create stable, self-contained visualizations for their own single-cell datasets. Learn more at https://cells.ucsc.edu.Supplementary informationSupplementary data are available at Bioinformatics online.

Microglia are resident macrophages in the brain that emerge in early development and respond to the local environment by altering their molecular and phenotypic states. Fundamental questions about microglia diversity and function during development remain unanswered because we lack experimental strategies to interrogate their interactions with other cell types and responses to perturbations ex vivo. We compared human microglia states across culture models, including cultured primary and pluripotent stem cell-derived microglia. We developed a "report card" of gene expression signatures across these distinct models to facilitate characterization of their responses across experimental models, perturbations, and disease conditions. Xenotransplantation of human microglia into cerebral organoids allowed us to characterize key transcriptional programs of developing microglia in vitro and reveal that microglia induce transcriptional changes in neural stem cells and decrease interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.

Objective.Neural activity represents a functional readout of neurons that is increasingly important to monitor in a wide range of experiments. Extracellular recordings have emerged as a powerful technique for measuring neural activity because these methods do not lead to the destruction or degradation of the cells being measured. Current approaches to electrophysiology have a low throughput of experiments due to manual supervision and expensive equipment. This bottleneck limits broader inferences that can be achieved with numerous long-term recorded samples.Approach.We developed Piphys, an inexpensive open source neurophysiological recording platform that consists of both hardware and software. It is easily accessed and controlled via a standard web interface through Internet of Things (IoT) protocols.Main results.We used a Raspberry Pi as the primary processing device along with an Intan bioamplifier. We designed a hardware expansion circuit board and software to enable voltage sampling and user interaction. This standalone system was validated with primary human neurons, showing reliability in collecting neural activity in near real-time.Significance.The hardware modules and cloud software allow for remote control of neural recording experiments as well as horizontal scalability, enabling long-term observations of development, organization, and neural activity at scale.

The human brain is subdivided into distinct anatomical structures, including the neocortex, which in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish early brain regions and cortical areas, but how early patterns result in finer and more discrete spatial differences remains poorly understood1. Here we use single-cell RNA sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Within the neocortex, we find that early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization approach, we find that laminar gene-expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene-expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.

During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscape1. In the developing brain, cell fate specification and topographic identity are important for defining cell identity2 and confer selective vulnerabilities to neurodevelopmental disorders3. Here, to identify cell-type-specific chromatin accessibility patterns in the developing human brain, we used a single-cell assay for transposase accessibility by sequencing (scATAC-seq) in primary tissue samples from the human forebrain. We applied unbiased analyses to identify genomic loci that undergo extensive cell-type- and brain-region-specific changes in accessibility during neurogenesis, and an integrative analysis to predict cell-type-specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell-type-specific enhancer accessibility patterns but lack many cell-type-specific open chromatin regions that are found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed unexpected diversity among neural progenitor cells in the cerebral cortex and implicated retinoic acid signalling in the specification of neuronal lineage identity in the prefrontal cortex. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development.

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.

A low cost remote imaging platform for biological applications was developed.The "Picroscope" is a device that allows the user to perform longitudinalimaging studies on multi-well cell culture plates. Here we present the networkarchitecture and software used to facilitate communication between moduleswithin the device as well as external cloud services. A web based console wascreated to control the device and view experiment results. Post processingtools were developed to analyze captured data in the cloud. The result is aplatform for controlling biological experiments from outside the lab.

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.

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods.

Background and Purpose: The management of unruptured intracranial aneurysms remains controversial. The decisions to treat are heavily informed by estimated risk of bleeding. However, these estimates are imprecise, and better methods for stratifying the risk or tailoring treatment strategy are badly needed. Here, we demonstrate an initial proof-of-principle concept for endovascular biopsy to identify the key molecular pathways and gene expression changes associated with aneurysm formation. We couple this technique with single cell RNA sequencing (scRNAseq) to develop a roadmap of the pathogenic changes of a dolichoectatic vertebrobasilar aneurysm in a patient with polyarteritis nodosa. Methods: Endovascular biopsy and fluorescence activated cell sorting was used to isolate the viable endothelial cells (ECs) using the established techniques. A single cell RNA sequencing (scRNAseq) was then performed on 24 aneurysmal ECs and 23 patient-matched non-aneurysmal ECs. An integrated panel of bioinformatic tools was applied to determine the differential gene expression, enriched signaling pathways, and cell subpopulations hypothesized to drive disease pathogenesis. Results: We identify a subset of 7 (29%) aneurysm-specific ECs with a distinct gene expression signature not found in the patient-matched control ECs. A gene set enrichment analysis identified these ECs to have increased the expression of genes regulating the leukocyte-endothelial cell adhesion, major histocompatibility complex (MHC) class I, T cell receptor recycling, tumor necrosis factor alpha (TNFα) response, and interferon gamma signaling. A histopathologic analysis of a different intracranial aneurysm that was later resected yielded a diagnosis of polyarteritis nodosa and positive staining for TNFα. Conclusions: We demonstrate feasibility of applying scRNAseq to the endovascular biopsy samples and identify a subpopulation of ECs associated with cerebral aneurysm in polyarteritis nodosa. Endovascular biopsy may be a safe method for deriving insight into the disease pathogenesis and tailoring the personalized treatment approaches to intracranial aneurysms.

Innovations in organoid-based models of human tissues have made them an exciting experimental platform for studying development and disease. However, these models require systematic benchmarking against primary tissue to establish their value. We discuss key parameters that impact the utility of organoid models, primarily focusing on cerebral organoids as examples.

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.

Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex1,2. However, the fidelity of organoid models remains unclear3-5. Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development.

The cerebrovasculature is essential to brain health and is tasked with ensuring adequate delivery of oxygen and metabolic precursors to ensure normal neurologic function. This is coordinated through a dynamic, multi-directional cellular interplay between vascular, neuronal, and glial cells. Molecular exchanges across the blood-brain barrier or the close matching of regional blood flow with brain activation are not uniformly assigned to arteries, capillaries, and veins. Evidence has supported functional segmentation of the brain vasculature. This is achieved in part through morphologic or transcriptional heterogeneity of brain vascular cells-including endothelium, pericytes, and vascular smooth muscle. Advances with single cell genomic technologies have shown increasing cell complexity of the brain vasculature identifying previously unknown cell types and further subclassifying transcriptional diversity in cardinal vascular cell types. Cell-type specific molecular transitions or zonations have been identified. In this review, we summarize emerging evidence for the expanding vascular cell diversity in the brain and how this may provide a cellular basis for functional segmentation along the arterial-venous axis.

Abstract Gene expression differs between cell types and regions within complex tissues such as the developing brain. To discover regulatory elements underlying this specificity, we generated genome-wide maps of chromatin accessibility in eleven anatomically-defined regions of the developing human telencephalon, including upper and deep layers of the prefrontal cortex. We predicted a subset of open chromatin regions (18%) that are most likely to be active enhancers, many of which are dynamic with 26% differing between early and late mid-gestation and 28% present in only one brain region. These region-specific predicted regulatory elements (pREs) are enriched proximal to genes with expression differences across regions and developmental stages and harbor distinct sequence motifs that suggest potential upstream regulators of regional and temporal transcription. We leverage this atlas to identify regulators of genes associated with autism spectrum disorder (ASD) including an enhancer of BCL11A , validated in mouse, and two functional de novo mutations in individuals with ASD in an enhancer of SLC6A1 , validated in neuroblastoma cells. These applications demonstrate the utility of this atlas for decoding neurodevelopmental gene regulation in health and disease. Summary 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 eleven dissected regions of the mid-gestation human telencephalon, including 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 regional differences in chromatin accessibility, including many specific to one brain region, and were correlated with gene expression differences across the same regions and gestational ages. pREs allowed us to map neurodevelopmental disorder risk genes to developing telencephalic regions, and we identified three functional de novo noncoding variants in pREs that alter enhancer function. In addition, transgenic experiments in mouse validated enhancer activity for a pRE proximal to BCL11A , showing how this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.

Neuroserpin is a serine-protease inhibitor mainly expressed in the CNS and involved in the inhibition of the proteolytic cascade. Animal models confirmed its neuroprotective role in perinatal hypoxia-ischaemia and adult stroke. Although neuroserpin may be a potential therapeutic target in the treatment of the aforementioned conditions, there is still no information in the literature on its distribution during human brain development. The present study provides a detailed description of the changing spatiotemporal patterns of neuroserpin focusing on physiological human brain development. Five stages were distinguished within our examined age range which spanned from the 7th gestational week until adulthood. In particular, subplate and deep cortical plate neurons were identified as the main sources of neuroserpin production between the 25th gestational week and the first postnatal month. Our immunohistochemical findings were substantiated by single cell RNA sequencing data showing specific neuronal and glial cell types expressing neuroserpin. The characterization of neuroserpin expression during physiological human brain development is essential for forthcoming studies which will explore its involvement in pathological conditions, such as perinatal hypoxia-ischaemia and adult stroke in human.

Adult cortical areas consist of specialized cell types and circuits that support unique higher-order cognitive functions. How this regional diversity develops from an initially uniform neuroepithelium has been the subject of decades of seminal research, and emerging technologies, including single-cell transcriptomics, provide a new perspective on area-specific molecular diversity. Here, we review the early developmental processes that underlie cortical arealization, including both cortex intrinsic and extrinsic mechanisms as embodied by the protomap and protocortex hypotheses, respectively. We propose an integrated model of serial homology whereby intrinsic genetic programs and local factors establish early transcriptomic differences between excitatory neurons destined to give rise to broad "proto-regions," and activity-dependent mechanisms lead to progressive refinement and formation of sharp boundaries between functional areas. Finally, we explore the potential of these basic developmental processes to inform our understanding of the emergence of functional neural networks and circuit abnormalities in neurodevelopmental disorders.

Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes.

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.

Mafb and c-Maf transcription factor (TF) expression is enriched in medial ganglionic eminence (MGE) lineages, beginning in late-secondary progenitors and continuing into mature parvalbumin (PV+) and somatostatin (SST+) interneurons. However, the functions of Maf TFs in MGE development remain to be elucidated. Herein, Mafb and c-Maf were conditionally deleted, alone and together, in the MGE and its lineages. Analyses of Maf mutant mice revealed redundant functions of Mafb and c-Maf in secondary MGE progenitors, where they repress the generation of SST+ cortical and hippocampal interneurons. By contrast, Mafb and c-Maf have distinct roles in postnatal cortical interneuron (CIN) morphological maturation, synaptogenesis, and cortical circuit integration. Thus, Mafb and c-Maf have redundant and opposing functions at different steps in CIN development.

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 (

MicroRNAs (miRNAs) regulate many cellular events during brain development by interacting with hundreds of mRNA transcripts. However, miRNAs operate nonuniformly upon the transcriptional profile with an as yet unknown logic. Shortcomings in defining miRNA-mRNA networks include limited knowledge of in vivo miRNA targets and their abundance in single cells. By combining multiple complementary approaches, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation with an antibody to AGO2 (AGO2-HITS-CLIP), single-cell profiling and computational analyses using bipartite and coexpression networks, we show that miRNA-mRNA interactions operate as functional modules that often correspond to cell-type identities and undergo dynamic transitions during brain development. These networks are highly dynamic during development and over the course of evolution. One such interaction is between radial-glia-enriched ORC4 and miR-2115, a great-ape-specific miRNA, which appears to control radial glia proliferation rates during human brain development.

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

Systematic analyses of spatiotemporal gene expression trajectories during organogenesis have been challenging because diverse cell types at different stages of maturation and differentiation coexist in the emerging tissues. We identified discrete cell types as well as temporally and spatially restricted trajectories of radial glia maturation and neurogenesis in developing human telencephalon. These lineage-specific trajectories reveal the expression of neurogenic transcription factors in early radial glia and enriched activation of mammalian target of rapamycin signaling in outer radial glia. Across cortical areas, modest transcriptional differences among radial glia cascade into robust typological distinctions among maturing neurons. Together, our results support a mixed model of topographical, typological, and temporal hierarchies governing cell-type diversity in the developing human telencephalon, including distinct excitatory lineages emerging in rostral and caudal cerebral cortex.

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy, and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study, to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology, we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging, immunostaining, and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells, accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia, a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study, therefore, deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

The classic view of cortical development, embodied in the radial unit hypothesis, highlights the ventricular radial glia (vRG) scaffold as a key architectonic feature of the developing neocortex. The scaffold includes continuous fibers spanning the thickness of the developing cortex during neurogenesis across mammals. However, we find that in humans, the scaffold transforms into a physically discontinuous structure during the transition from infragranular to supragranular neuron production. As a consequence of this transformation, supragranular layer neurons arrive at their terminal positions in the cortical plate along outer radial glia (oRG) cell fibers. In parallel, the radial glia that contact the ventricle develop distinct gene expression profile and "truncated" morphology. We propose a supragranular layer expansion hypothesis that posits a deterministic role of oRG cells in the radial and tangential expansion of supragranular layers in primates, with implications for patterns of neuronal migration, area patterning, and cortical folding.

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.

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.