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.

Lineage-specific epigenomic changes during human corticogenesis have remained elusive due to challenges with sample availability and tissue heterogeneity. For example, previous studies used single-cell RNA sequencing to identify at least nine major cell types and up to 26 distinct subtypes in the dorsal cortex alone 1 , 2 . Here, we characterize cell type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational human cortex samples. We show that chromatin interactions underlie multiple aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell type-specific manner. In addition, promoters with significantly increased levels of chromatin interactivity, termed super interactive promoters, are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a novel technique integrating immunostaining, CRISPRi, RNAscope, and image analysis for validating cell type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our study presents the first cell type-specific characterization of 3D epigenomes in the developing human cortex, advancing our understanding of gene regulation and lineage specification during this critical developmental window.

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.

Graphical Abstract Highlights d Multimodal analysis differentiates cells beyond transcriptomic classification d Single-cell analysis links stimulus-induced calcium elevations to transcriptomes d Cell-type-specific responses to neurotransmitters are associated with maturation d Serotonergic signaling in human radial glia promotes radial fiber formation

The human brain is subdivided into distinct anatomical structures. The neocortex, one of these structures, enables higher-order sensory, associative, and cognitive functions, and in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish an early blueprint for the specification of brain regions and cortical areas. Furthermore, recent studies have uncovered distinct transcriptomic signatures between opposing poles of the developing neocortex1. However, how early, broad developmental patterns result in finer and more discrete spatial differences across the adult human brain remains poorly understood2. Here, we use single-cell RNA-sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Our data reveal that distinct cell subtypes are predominantly brain-structure specific. Within the neocortex, we find that even 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 (smFISH) approach, we validated the expression pattern of area-specific neuronal genes and also discover 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.

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.

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. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion 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 primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.

The human cortex is comprised of diverse cell types that emerge from an initially uniform neuroepithelium that, following neural tube closure, gives rise to radial glia, the neural stem cells of the cortex. Radial glia initially reside in the ventricular zone of the cortex, and contribute to cortical expansion which is particularly pronounced in the human compared to other mammals and non-human primates. To characterize the molecular signatures of cellular subtypes that may exist at the earliest stages of neurogenesis we performed single-cell RNA-sequencing across regions of the developing human brain. We observe similar progenitor programs across brain regions that each express region-specific transcription factors. In the telencephalon, we identify nine progenitor populations, suggesting more heterogeneity among neuroepithelial cells and radial glia than previously described, including a highly prevalent mesenchymal-like population that disappears after the onset of neurogenesis. Using velocity analysis, we find that genes implicated in various neurodevelopmental diseases drive the specification and maintenance of neuroepithelial cells and radial glia. Comparison of these progenitor populations to corresponding stages of mouse development identifies two progenitor clusters that are unique to the early stages of human cortical development. Organoid systems display a low fidelity to neuroepithelial and early radial glia cell types, but this improves as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.The human brain consists of billions of cells across several functionally interconnected structures that emerge from the neuroectoderm. Many of these structures are substantially expanded or distinct compared to other mammals 1 , particularly the cerebral cortex, the outermost layer of the human brain responsible for perception and cognition. These differences emerge at developmental stages prior to birth 2 , and thus exploring the cell types in the developing human brain is essential to better characterize how cell types across the brain are generated, how they may be impacted during the emergence of neurodevelopmental disorders, and how human neural stem cells can be directed to specific cell types for modeling or treatment purposes. The brain exponentially increases in size after the neural tube closes 3,4 . Later in development, across brain regions, a series of similar neurogenic and gliogenic processes gives rise to the constituent cell types 5 . However, at the molecular level, the sequence of events that leads to the emergence of these progenitor cells early in development is much less well understood. In order to identify cell types and trajectories that lay the foundation for the development of the human brain, we performed single-cell RNA sequencing (scRNA-seq) using the droplet-based 10X Genomics Chromium platform on ten individuals during the first trimester of human development, spanning Carnegie Stages (CS) ...