Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Large-scale neurophysiology and single-cell profiling in human neuroscience.

Advances in large-scale single-unit human neurophysiology, single-cell RNA sequencing, spatial transcriptomics and long-term ex vivo tissue culture of surgically resected human brain tissue have provided an unprecedented opportunity to study human neuroscience. In this Perspective, we describe the development of these paradigms, including Neuropixels and recent brain-cell atlas efforts, and discuss how their convergence will further investigations into the cellular underpinnings of network-level activity in the human brain. Specifically, we introduce a workflow in which functionally mapped samples of human brain tissue resected during awake brain surgery can be cultured ex vivo for multi-modal cellular and functional profiling. We then explore how advances in human neuroscience will affect clinical practice, and conclude by discussing societal and ethical implications to consider. Potential findings from the field of human neuroscience will be vast, ranging from insights into human neurodiversity and evolution to providing cell-type-specific access to study and manipulate diseased circuits in pathology. This Perspective aims to provide a unifying framework for the field of human neuroscience as we welcome an exciting era for understanding the functional cytoarchitecture of the human brain.

A cross-species proteomic map reveals neoteny of human synapse development.

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.

The development and evolution of inhibitory neurons in primate cerebrum.

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.

ß-Adrenergic Signaling Promotes Morphological Maturation of Astrocytes in Female Mice.

Astrocytes play essential roles in the developing nervous system, including supporting synapse function. These astrocyte support functions emerge coincident with brain maturation and may be tailored in a region-specific manner. For example, gray matter astrocytes have elaborate synapse-associated processes and are morphologically and molecularly distinct from white matter astrocytes. This raises the question of whether there are unique environmental cues that promote gray matter astrocyte identity and synaptogenic function. We previously identified adrenergic receptors as preferentially enriched in developing gray versus white matter astrocytes, suggesting that noradrenergic signaling could be a cue that promotes the functional maturation of gray matter astrocytes. We first characterized noradrenergic projections during postnatal brain development in mouse and human, finding that process density was higher in the gray matter and increased concurrently with astrocyte maturation. RNA sequencing revealed that astrocytes in both species expressed α- and ß-adrenergic receptors. We found that stimulation of ß-adrenergic receptors increased primary branching of rodent astrocytes in vitro Conversely, astrocyte-conditional knockout of the ß1-adrenergic receptor reduced the size of gray matter astrocytes and led to dysregulated sensorimotor integration in female mice. These studies suggest that adrenergic signaling to developing astrocytes impacts their morphology and has implications for adult behavior, particularly in female animals. More broadly, they demonstrate a mechanism through which environmental cues impact astrocyte development. Given the key roles of norepinephrine in brain states, such as arousal, stress, and learning, these findings could prompt further inquiry into how developmental stressors impact astrocyte development and adult brain function.SIGNIFICANCE STATEMENT This study demonstrates a role for noradrenergic signaling in the development of gray matter astrocytes. We provide new evidence that the ß1-adrenergic receptor is robustly expressed by both mouse and human astrocytes, and that conditional KO of the ß1-adrenergic receptor from female mouse astrocytes impairs gray matter astrocyte maturation. Moreover, female conditional KO mice exhibit behavioral deficits in two paradigms that test sensorimotor function. Given the emerging interest in moving beyond RNA sequencing to probe specific pathways that underlie astrocyte heterogeneity, this study provides a foundation for future investigation into the effect of noradrenergic signaling on astrocyte functions in conditions where noradrenergic signaling is altered, such as stress, arousal, and learning.

Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults.

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.

Interneuron Origins in the Embryonic Porcine Medial Ganglionic Eminence.

Interneurons contribute to the complexity of neural circuits and maintenance of normal brain function. Rodent interneurons originate in embryonic ganglionic eminences, but developmental origins in other species are less understood. Here, we show that transcription factor expression patterns in porcine embryonic subpallium are similar to rodents, delineating a distinct medial ganglionic eminence (MGE) progenitor domain. On the basis of Nkx2.1, Lhx6, and Dlx2 expression, in vitro differentiation into neurons expressing GABA, and robust migratory capacity in explant assays, we propose that cortical and hippocampal interneurons originate from a porcine MGE region. Following xenotransplantation into adult male and female rat hippocampus, we further demonstrate that porcine MGE progenitors, like those from rodents, migrate and differentiate into morphologically distinct interneurons expressing GABA. Our findings reveal that basic rules for interneuron development are conserved across species, and that porcine embryonic MGE progenitors could serve as a valuable source for interneuron-based xenotransplantation therapies.SIGNIFICANCE STATEMENT Here we demonstrate that porcine medial ganglionic eminence, like rodents, exhibit a distinct transcriptional and interneuron-specific antibody profile, in vitro migratory capacity and are amenable to xenotransplantation. This is the first comprehensive examination of embryonic interneuron origins in the pig; and because a rich neurodevelopmental literature on embryonic mouse medial ganglionic eminence exists (with some additional characterizations in other species, e.g., monkey and human), our work allows direct neurodevelopmental comparisons with this literature.

Positive Controls in Adults and Children Support That Very Few, If Any, New Neurons Are Born in the Adult Human Hippocampus.

Adult hippocampal neurogenesis was originally discovered in rodents. Subsequent studies identified the adult neural stem cells and found important links between adult neurogenesis and plasticity, behavior, and disease. However, whether new neurons are produced in the human dentate gyrus (DG) during healthy aging is still debated. We and others readily observe proliferating neural progenitors in the infant hippocampus near immature cells expressing doublecortin (DCX), but the number of such cells decreases in children and few, if any, are present in adults. Recent investigations using dual antigen retrieval find many cells stained by DCX antibodies in adult human DG. This has been interpreted as evidence for high rates of adult neurogenesis, even at older ages. However, most of these DCX-labeled cells have mature morphology. Furthermore, studies in the adult human DG have not found a germinal region containing dividing progenitor cells. In this Dual Perspectives article, we show that dual antigen retrieval is not required for the detection of DCX in multiple human brain regions of infants or adults. We review prior studies and present new data showing that DCX is not uniquely expressed by newly born neurons: DCX is present in adult amygdala, entorhinal and parahippocampal cortex neurons despite being absent in the neighboring DG. Analysis of available RNA-sequencing datasets supports the view that DG neurogenesis is rare or absent in the adult human brain. To resolve the conflicting interpretations in humans, it is necessary to identify and visualize dividing neuronal precursors or develop new methods to evaluate the age of a neuron at the single-cell level.

Implications of Extended Inhibitory Neuron Development.

A prolonged developmental timeline for GABA (γ-aminobutyric acid)-expressing inhibitory neurons (GABAergic interneurons) is an amplified trait in larger, gyrencephalic animals. In several species, the generation, migration, and maturation of interneurons take place over several months, in some cases persisting after birth. The late integration of GABAergic interneurons occurs in a region-specific pattern, especially during the early postnatal period. These changes can contribute to the formation of functional connectivity and plasticity, especially in the cortical regions responsible for higher cognitive tasks. In this review, we discuss GABAergic interneuron development in the late gestational and postnatal forebrain. We propose the protracted development of interneurons at each stage (neurogenesis, neuronal migration, and network integration), as a mechanism for increased complexity and cognitive flexibility in larger, gyrencephalic brains. This developmental feature of interneurons also provides an avenue for environmental influences to shape neural circuit formation.

Secretagogin is Expressed by Developing Neocortical GABAergic Neurons in Humans but not Mice and Increases Neurite Arbor Size and Complexity.

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.

Intrinsically determined cell death of developing cortical interneurons.

Cortical inhibitory circuits are formed by γ-aminobutyric acid (GABA)-secreting interneurons, a cell population that originates far from the cerebral cortex in the embryonic ventral forebrain. Given their distant developmental origins, it is intriguing how the number of cortical interneurons is ultimately determined. One possibility, suggested by the neurotrophic hypothesis, is that cortical interneurons are overproduced, and then after their migration into cortex the excess interneurons are eliminated through a competition for extrinsically derived trophic signals. Here we characterize the developmental cell death of mouse cortical interneurons in vivo, in vitro and after transplantation. We found that 40% of developing cortical interneurons were eliminated through Bax (Bcl-2-associated X)-dependent apoptosis during postnatal life. When cultured in vitro or transplanted into the cortex, interneuron precursors died at a cellular age similar to that at which endogenous interneurons died during normal development. Over transplant sizes that varied 200-fold, a constant fraction of the transplanted population underwent cell death. The death of transplanted neurons was not affected by the cell-autonomous disruption of TrkB (tropomyosin kinase receptor B), the main neurotrophin receptor expressed by neurons of the central nervous system. Transplantation expanded the cortical interneuron population by up to 35%, but the frequency of inhibitory synaptic events did not scale with the number of transplanted interneurons. Taken together, our findings indicate that interneuron cell death is determined intrinsically, either cell-autonomously or through a population-autonomous competition for survival signals derived from other interneurons.

Cell-Type Specificity of Mosaic Chromosome 1q Gain Resolved by snRNA-seq in a Case of Epilepsy With Hyaline Protoplasmic Astrocytopathy.

Objectives: Mosaic gain of chromosome 1q (chr1q) has been associated with malformation of cortical development (MCD) and epilepsy. Hyaline protoplasmic astrocytopathy (HPA) is a rare neuropathologic finding seen in cases of epilepsy with MCD. The cell-type specificity of mosaic chr1q gain in the brain and the molecular signatures of HPA are unknown. Methods: We present the case of a child with pharmacoresistant epilepsy who underwent epileptic focus resections at age 3 and 5 years and was found to have mosaic chr1q gain and HPA. We performed single-nuclei RNA sequencing (snRNA-seq) of brain tissue from the second resection. Results: snRNA-seq showed increased expression of chr1q genes specifically in subsets of neurons and astrocytes. Differentially expressed genes associated with inferred chr1q gain included AKT3 and genes associated with cell adhesion or migration. A subpopulation of astrocytes demonstrated marked enrichment for synapse-associated transcripts, possibly linked to the astrocytic inclusions observed in HPA. Discussion: snRNA-seq may be used to infer the cell-type specificity of mosaic chromosomal copy number changes and identify associated gene expression alterations, which in the case of chr1q gain may involve aberrations in cell migration. Future studies using spatial profiling could yield further insights on the molecular signatures of HPA.

Cell type specificity of mosaic chromosome 1q gain resolved by snRNA-seq in a case of epilepsy with hyaline protoplasmic astrocytopathy.

Introduction: Mosaic gain of chromosome 1q (chr1q) has been associated with malformation of cortical development (MCD) and epilepsy. Hyaline protoplasmic astrocytopathy (HPA) is a rare neuropathological finding seen in cases of epilepsy with MCD. The cell-type specificity of mosaic chr1q gain in the brain and the molecular signatures of HPA are unknown. Methods: We present a child with pharmacoresistant epilepsy who underwent epileptic focus resections at age 3 and 5 years and was found to have mosaic chr1q gain and HPA. We performed single-nuclei RNA-sequencing (snRNA-seq) of brain tissue from the second resection. Results: snRNA-seq showed increased expression of chr1q genes specifically in subsets of neurons and astrocytes. Differentially expressed genes associated with inferred chr1q gain included AKT3 and genes associated with cell adhesion or migration. A subpopulation of astrocytes demonstrated marked enrichment for synapse-associated transcripts, possibly linked to the astrocytic inclusions observed in HPA. Discussion: snRNA-seq may be used to infer the cell type-specificity of mosaic chromosomal copy number changes and identify associated gene expression alterations, which in the case of chr1q gain may involve aberrations in cell migration. Future studies using spatial profiling could yield further insights on the molecular signatures of HPA.

Comment on "Impact of neurodegenerative diseases on human adult hippocampal neurogenesis".

Terreros-Roncal et al. investigated the impacts of human neurodegeneration on immunostainings assumed to be associated with neurogenesis. However, the study provides no evidence that putative proliferating cells are linked to neurogenesis, that multipolar nestin+ astrocytes are progenitors, or that mature-looking doublecortin+ neurons are adult-born. Their histology-marker expression differs from what is observed in species where adult hippocampal neurogenesis is well documented.

Amplification of human interneuron progenitors promotes brain tumors and neurological defects.

Evolutionary development of the human brain is characterized by the expansion of various brain regions. Here, we show that developmental processes specific to humans are responsible for malformations of cortical development (MCDs), which result in developmental delay and epilepsy in children. We generated a human cerebral organoid model for tuberous sclerosis complex (TSC) and identified a specific neural stem cell type, caudal late interneuron progenitor (CLIP) cells. In TSC, CLIP cells over-proliferate, generating excessive interneurons, brain tumors, and cortical malformations. Epidermal growth factor receptor inhibition reduces tumor burden, identifying potential treatment options for TSC and related disorders. The identification of CLIP cells reveals the extended interneuron generation in the human brain as a vulnerability for disease. In addition, this work demonstrates that analyzing MCDs can reveal fundamental insights into human-specific aspects of brain development.

Nests of dividing neuroblasts sustain interneuron production for the developing human brain.

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.

Maf and Mafb control mouse pallial interneuron fate and maturation through neuropsychiatric disease gene regulation.

​Maf (c-Maf) and Mafb transcription factors (TFs) have compensatory roles in repressing somatostatin (SST+) interneuron (IN) production in medial ganglionic eminence (MGE) secondary progenitors in mice. Maf and Mafb conditional deletion (cDKO) decreases the survival of MGE-derived cortical interneurons (CINs) and changes their physiological properties. Herein, we show that (1) Mef2c and Snap25 are positively regulated by Maf and Mafb to drive IN morphological maturation; (2) Maf and Mafb promote Mef2c expression which specifies parvalbumin (PV+) INs; (3) Elmo1, Igfbp4 and Mef2c are candidate markers of immature PV+ hippocampal INs (HIN). Furthermore, Maf/Mafb neonatal cDKOs have decreased CINs and increased HINs, that express Pnoc, an HIN specific marker. Our findings not only elucidate key gene targets of Maf and Mafb that control IN development, but also identify for the first time TFs that differentially regulate CIN vs. HIN production.

Reciprocal Interaction between Vascular Filopodia and Neural Stem Cells Shapes Neurogenesis in the Ventral Telencephalon.

Angiogenesis and neurogenesis are tightly coupled during embryonic brain development. However, little is known about how these two processes interact. We show that nascent blood vessels actively contact dividing neural stem cells by endothelial filopodia in the ventricular zone (VZ) of the murine ventral telencephalon; this association is conserved in the human ventral VZ. Using mouse mutants with altered vascular filopodia density, we show that this interaction leads to prolonged cell cycle of apical neural progenitors (ANPs) and favors early neuronal differentiation. Interestingly, pharmacological experiments reveal that ANPs induce vascular filopodia formation by upregulating vascular endothelial growth factor (VEGF)-A in a cell-cycle-dependent manner. This mutual relationship between vascular filopodia and ANPs works as a self-regulatory system that senses ANP proliferation rates and rapidly adjusts neuronal production levels. Our findings indicate a function of vascular filopodia in fine-tuning neural stem cell behavior, which is the basis for proper brain development.

Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map.

Although the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. In the present study, we developed a high-content pipeline, the large-area spatial transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in the early postnatal cortex, mostly persisted in adult mouse and human cortex. Single-cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae.

Cortical Malformations: Lessons in Human Brain Development.

Creating a functional cerebral cortex requires a series of complex and well-coordinated developmental steps. These steps have evolved across species with the emergence of cortical gyrification and coincided with more complex behaviors. The presence of diverse progenitor cells, a protracted timeline for neuronal migration and maturation, and diverse neuronal types are developmental features that have emerged in the gyrated cortex. These factors could explain how the human brain has expanded in size and complexity. However, their complex nature also renders new avenues of vulnerability by providing additional cell types that could contribute to disease and longer time windows that could impact the composition and organization of the cortical circuit. We aim to discuss the unique developmental steps observed in human corticogenesis and propose how disruption of these species-unique processes could lead to malformations of cortical development.