Type-I-interferon-responsive microglia shape cortical development and behavior.

Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional states have been defined, but their functional significance is unclear. Here, we identify a type I interferon (IFN-I)-responsive microglial state in the developing somatosensory cortex (postnatal day 5) that is actively engulfing whole neurons. This population expands during cortical remodeling induced by partial whisker deprivation. Global or microglial-specific loss of the IFN-I receptor resulted in microglia with phagolysosomal dysfunction and an accumulation of neurons with nuclear DNA damage. IFN-I gain of function increased neuronal engulfment by microglia in both mouse and zebrafish and restricted the accumulation of DNA-damaged neurons. Finally, IFN-I deficiency resulted in excess cortical excitatory neurons and tactile hypersensitivity. These data define a role for neuron-engulfing microglia during a critical window of brain development and reveal homeostatic functions of a canonical antiviral signaling pathway in the brain.

Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex.

Realizing the full utility of brain organoids to study human development requires understanding whether organoids precisely replicate endogenous cellular and molecular events, particularly since acquisition of cell identity in organoids can be impaired by abnormal metabolic states. We present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, from generation of neural progenitors through production of differentiated neuronal and glial subtypes. We show that processes of cellular diversification correlate closely to endogenous ones, irrespective of metabolic state, empowering the use of this atlas to study human fate specification. We define longitudinal molecular trajectories of cortical cell types during organoid development, identify genes with predicted human-specific roles in lineage establishment, and uncover early transcriptional diversity of human callosal neurons. The findings validate this comprehensive atlas of human corticogenesis in vitro as a resource to prime investigation into the mechanisms of human cortical development.

Genetic Control of Expression and Splicing in Developing Human Brain Informs Disease Mechanisms.

Tissue-specific regulatory regions harbor substantial genetic risk for disease. Because brain development is a critical epoch for neuropsychiatric disease susceptibility, we characterized the genetic control of the transcriptome in 201 mid-gestational human brains, identifying 7,962 expression quantitative trait loci (eQTL) and 4,635 spliceQTL (sQTL), including several thousand prenatal-specific regulatory regions. We show that significant genetic liability for neuropsychiatric disease lies within prenatal eQTL and sQTL. Integration of eQTL and sQTL with genome-wide association studies (GWAS) via transcriptome-wide association identified dozens of novel candidate risk genes, highlighting shared and stage-specific mechanisms in schizophrenia (SCZ). Gene network analysis revealed that SCZ and autism spectrum disorder (ASD) affect distinct developmental gene co-expression modules. Yet, in each disorder, common and rare genetic variation converges within modules, which in ASD implicates superficial cortical neurons. More broadly, these data, available as a web browser and our analyses, demonstrate the genetic mechanisms by which developmental events have a widespread influence on adult anatomical and behavioral phenotypes.

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

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.

Progenitor Hyperpolarization Regulates the Sequential Generation of Neuronal Subtypes in the Developing Neocortex.

During corticogenesis, ventricular zone progenitors sequentially generate distinct subtypes of neurons, accounting for the diversity of neocortical cells and the circuits they form. While activity-dependent processes are critical for the differentiation and circuit assembly of postmitotic neurons, how bioelectrical processes affect nonexcitable cells, such as progenitors, remains largely unknown. Here, we reveal that, in the developing mouse neocortex, ventricular zone progenitors become more hyperpolarized as they generate successive subtypes of neurons. Experimental in vivo hyperpolarization shifted the transcriptional programs and division modes of these progenitors to a later developmental status, with precocious generation of intermediate progenitors and a forward shift in the laminar, molecular, morphological, and circuit features of their neuronal progeny. These effects occurred through inhibition of the Wnt-beta-catenin signaling pathway by hyperpolarization. Thus, during corticogenesis, bioelectric membrane properties are permissive for specific molecular pathways to coordinate the temporal progression of progenitor developmental programs and thus neocortical neuron diversity.

Multiscale 3D Genome Rewiring during Mouse Neural Development.

Chromosome conformation capture technologies have revealed important insights into genome folding. Yet, how spatial genome architecture is related to gene expression and cell fate remains unclear. We comprehensively mapped 3D chromatin organization during mouse neural differentiation in vitro and in vivo, generating the highest-resolution Hi-C maps available to date. We found that transcription is correlated with chromatin insulation and long-range interactions, but dCas9-mediated activation is insufficient for creating TAD boundaries de novo. Additionally, we discovered long-range contacts between gene bodies of exon-rich, active genes in all cell types. During neural differentiation, contacts between active TADs become less pronounced while inactive TADs interact more strongly. An extensive Polycomb network in stem cells is disrupted, while dynamic interactions between neural transcription factors appear in vivo. Finally, cell type-specific enhancer-promoter contacts are established concomitant to gene expression. This work shows that multiple factors influence the dynamics of chromatin interactions in development.

BAF subunit switching regulates chromatin accessibility to control cell cycle exit in the developing mammalian cortex.

mSWI/SNF or BAF chromatin regulatory complexes are dosage-sensitive regulators of human neural development frequently mutated in autism spectrum disorders and intellectual disability. Cell cycle exit and differentiation of neural stem/progenitor cells is accompanied by BAF subunit switching to generate neuron-specific nBAF complexes. We manipulated the timing of BAF subunit exchange in vivo and found that early loss of the npBAF subunit BAF53a stalls the cell cycle to disrupt neurogenesis. Loss of BAF53a results in decreased chromatin accessibility at specific neural transcription factor binding sites, including the pioneer factors SOX2 and ASCL1, due to Polycomb accumulation. This results in repression of cell cycle genes, thereby blocking cell cycle progression and differentiation. Cell cycle block upon Baf53a deletion could be rescued by premature expression of the nBAF subunit BAF53b but not by other major drivers of proliferation or differentiation. WNT, EGF, bFGF, SOX2, c-MYC, or PAX6 all fail to maintain proliferation in the absence of BAF53a, highlighting a novel mechanism underlying neural progenitor cell cycle exit in the continued presence of extrinsic proliferative cues.

Phf21b imprints the spatiotemporal epigenetic switch essential for neural stem cell differentiation.

Cerebral cortical development in mammals involves a highly complex and organized set of events including the transition of neural stem and progenitor cells (NSCs) from proliferative to differentiative divisions to generate neurons. Despite progress, the spatiotemporal regulation of this proliferation-differentiation switch during neurogenesis and the upstream epigenetic triggers remain poorly known. Here we report a cortex-specific PHD finger protein, Phf21b, which is highly expressed in the neurogenic phase of cortical development and gets induced as NSCs begin to differentiate. Depletion of Phf21b in vivo inhibited neuronal differentiation as cortical progenitors lacking Phf21b were retained in the proliferative zones and underwent faster cell cycles. Mechanistically, Phf21b targets the regulatory regions of cell cycle promoting genes by virtue of its high affinity for monomethylated H3K4. Subsequently, Phf21b recruits the lysine-specific demethylase Lsd1 and histone deacetylase Hdac2, resulting in the simultaneous removal of monomethylation from H3K4 and acetylation from H3K27, respectively. Intriguingly, mutations in the Phf21b locus associate with depression and mental retardation in humans. Taken together, these findings establish how a precisely timed spatiotemporal expression of Phf21b creates an epigenetic program that triggers neural stem cell differentiation during cortical development.

Development and evolution of the primate neocortex from a progenitor cell perspective.

The generation of neurons in the developing neocortex is a major determinant of neocortex size. Crucially, the increase in cortical neuron numbers in the primate lineage, notably in the upper-layer neurons, contributes to increased cognitive abilities. Here, we review major evolutionary changes affecting the apical progenitors in the ventricular zone and focus on the key germinal zone constituting the foundation of neocortical neurogenesis in primates, the outer subventricular zone (OSVZ). We summarize characteristic features of the OSVZ and its key stem cell type, the basal (or outer) radial glia. Next, we concentrate on primate-specific and human-specific genes, expressed in OSVZ-progenitors, the ability of which to amplify these progenitors by targeting the regulation of the cell cycle ultimately underlies the evolutionary increase in upper-layer neurons. Finally, we address likely differences in neocortical development between present-day humans and Neanderthals that are based on human-specific amino acid substitutions in proteins operating in cortical progenitors.

Fetal brain response to maternal inflammation requires microglia.

In utero infection and maternal inflammation can adversely impact fetal brain development. Maternal systemic illness, even in the absence of direct fetal brain infection, is associated with an increased risk of neuropsychiatric disorders in affected offspring. The cell types mediating the fetal brain response to maternal inflammation are largely unknown, hindering the development of novel treatment strategies. Here, we show that microglia, the resident phagocytes of the brain, highly express receptors for relevant pathogens and cytokines throughout embryonic development. Using a rodent maternal immune activation (MIA) model in which polyinosinic:polycytidylic acid is injected into pregnant mice, we demonstrate long-lasting transcriptional changes in fetal microglia that persist into postnatal life. We find that MIA induces widespread gene expression changes in neuronal and non-neuronal cells; importantly, these responses are abolished by selective genetic deletion of microglia, indicating that microglia are required for the transcriptional response of other cortical cell types to MIA. These findings demonstrate that microglia play a crucial durable role in the fetal response to maternal inflammation, and should be explored as potential therapeutic cell targets.

A lissencephaly-associated BAIAP2 variant causes defects in neuronal migration during brain development.

Lissencephaly is a neurodevelopmental disorder characterized by a loss of brain surface convolutions caused by genetic variants that disrupt neuronal migration. However, the genetic origins of the disorder remain unidentified in nearly one-fifth of people with lissencephaly. Using whole-exome sequencing, we identified a de novo BAIAP2 variant, p.Arg29Trp, in an individual with lissencephaly with a posterior more severe than anterior (P>A) gradient, implicating BAIAP2 as a potential lissencephaly gene. Spatial transcriptome analysis in the developing mouse cortex revealed that Baiap2 is expressed in the cortical plate and intermediate zone in an anterior low to posterior high gradient. We next used in utero electroporation to explore the effects of the Baiap2 variant in the developing mouse cortex. We found that Baiap2 knockdown caused abnormalities in neuronal migration, morphogenesis and differentiation. Expression of the p.Arg29Trp variant failed to rescue the migration defect, suggesting a loss-of-function effect. Mechanistically, the variant interfered with the ability of BAIAP2 to localize to the cell membrane. These results suggest that the functions of BAIAP2 in the cytoskeleton, cell morphogenesis and migration are important for cortical development and for the pathogenesis of lissencephaly in humans.

Temporal and sequential transcriptional dynamics define lineage shifts in corticogenesis.

The cerebral cortex contains billions of neurons, and their disorganization or misspecification leads to neurodevelopmental disorders. Understanding how the plethora of projection neuron subtypes are generated by cortical neural stem cells (NSCs) is a major challenge. Here, we focused on elucidating the transcriptional landscape of murine embryonic NSCs, basal progenitors (BPs), and newborn neurons (NBNs) throughout cortical development. We uncover dynamic shifts in transcriptional space over time and heterogeneity within each progenitor population. We identified signature hallmarks of NSC, BP, and NBN clusters and predict active transcriptional nodes and networks that contribute to neural fate specification. We find that the expression of receptors, ligands, and downstream pathway components is highly dynamic over time and throughout the lineage implying differential responsiveness to signals. Thus, we provide an expansive compendium of gene expression during cortical development that will be an invaluable resource for studying neural developmental processes and neurodevelopmental disorders.

Notch directs telencephalic development and controls neocortical neuron fate determination by regulating microRNA levels.

The central nervous system contains a myriad of different cell types produced from multipotent neural progenitors. Neural progenitors acquire distinct cell identities depending on their spatial position, but they are also influenced by temporal cues to give rise to different cell populations over time. For instance, the progenitors of the cerebral neocortex generate different populations of excitatory projection neurons following a well-known sequence. The Notch signaling pathway plays crucial roles during this process, but the molecular mechanisms by which Notch impacts progenitor fate decisions have not been fully resolved. Here, we show that Notch signaling is essential for neocortical and hippocampal morphogenesis, and for the development of the corpus callosum and choroid plexus. Our data also indicate that, in the neocortex, Notch controls projection neuron fate determination through the regulation of two microRNA clusters that include let-7, miR-99a/100 and miR-125b. Our findings collectively suggest that balanced Notch signaling is crucial for telencephalic development and that the interplay between Notch and miRNAs is essential for the control of neocortical progenitor behaviors and neuron cell fate decisions.

Entosis implicates a new role for P53 in microcephaly pathogenesis, beyond apoptosis.

Entosis, a form of cell cannibalism, is a newly discovered pathogenic mechanism leading to the development of small brains, termed microcephaly, in which P53 activation was found to play a major role. Microcephaly with entosis, found in Pals1 mutant mice, displays P53 activation that promotes entosis and apoptotic cell death. This previously unappreciated pathogenic mechanism represents a novel cellular dynamic in dividing cortical progenitors which is responsible for cell loss. To date, various recent models of microcephaly have bolstered the importance of P53 activation in cell death leading to microcephaly. P53 activation caused by mitotic delay or DNA damage manifests apoptotic cell death which can be suppressed by P53 removal in these animal models. Such genetic studies attest P53 activation as quality control meant to eliminate genomically unfit cells with minimal involvement in the actual function of microcephaly associated genes. In this review, we summarize the known role of P53 activation in a variety of microcephaly models and introduce a novel mechanism wherein entotic cell cannibalism in neural progenitors is triggered by P53 activation.

Longitudinal mapping of the development of cortical thickness and surface area in rhesus macaques during the first three years.

Studying dynamic spatiotemporal patterns of early brain development in macaque monkeys is critical for understanding the cortical organization and evolution in humans, given the phylogenetic closeness between humans and macaques. However, due to huge challenges in the analysis of early brain Magnetic Resonance Imaging (MRI) data typically with extremely low contrast and dynamic imaging appearances, our knowledge of the early macaque cortical development remains scarce. To fill this critical gap, this paper characterizes the early developmental patterns of cortical thickness and surface area in rhesus macaques by leveraging advanced computing tools tailored for early developing brains based on a densely sampled longitudinal dataset with 140 rhesus macaque MRI scans seamlessly covering from birth to 36 mo of age. The average cortical thickness exhibits an inverted U-shaped trajectory with peak thickness at around 4.3 mo of age, which is remarkably in line with the age of peak thickness at 14 mo in humans, considering the around 3:1 age ratio of human to macaque. The total cortical surface area in macaques increases monotonically but with relatively lower expansions than in humans. The spatial distributions of thicker and thinner regions are quite consistent during development, with gyri having a thicker cortex than sulci. By 4 mo of age, over 81% of cortical vertices have reached their peaks in thickness, except for the insula and medial temporal cortices, while most cortical vertices keep expanding in surface area, except for the occipital cortex. These findings provide important insights into early brain development and evolution in primates.

Cortical activity emerges in region-specific patterns during early brain development.

The development of precise neural circuits in the brain requires spontaneous patterns of neural activity prior to functional maturation. In the rodent cerebral cortex, patchwork and wave patterns of activity develop in somatosensory and visual regions, respectively, and are present at birth. However, whether such activity patterns occur in noneutherian mammals, as well as when and how they arise during development, remain open questions relevant for understanding brain formation in health and disease. Since the onset of patterned cortical activity is challenging to study prenatally in eutherians, here we offer an approach in a minimally invasive manner using marsupial dunnarts, whose cortex forms postnatally. We discovered similar patchwork and travelling waves in the dunnart somatosensory and visual cortices at stage 27 (equivalent to newborn mice) and examined earlier stages of development to determine the onset of these patterns and how they first emerge. We observed that these patterns of activity emerge in a region-specific and sequential manner, becoming evident as early as stage 24 in somatosensory and stage 25 in visual cortices (equivalent to embryonic day 16 and 17, respectively, in mice), as cortical layers establish and thalamic axons innervate the cortex. In addition to sculpting synaptic connections of existing circuits, evolutionarily conserved patterns of neural activity could therefore help regulate other early events in cortical development.

Tubulin mutations in human neurodevelopmental disorders.

Mutations causing dysfunction of tubulins and microtubule-associated proteins, also known as tubulinopathies, are a group of recently described entities that lead to complex brain malformations. Anatomical and functional consequences of the disruption of tubulins include microcephaly, combined with abnormal corticogenesis due to impaired migration or lamination and abnormal growth cone dynamics of projecting and callosal axons. Key imaging features of tubulinopathies are characterized by three major patterns of malformations of cortical development (MCD): lissencephaly, microlissencephaly, and dysgyria. Additional distinctive MRI features include dysmorphism of the basal ganglia, midline commissural structure hypoplasia or agenesis, and cerebellar and brainstem hypoplasia. Tubulinopathies can be diagnosed as early as 21-24 gestational weeks using imaging and neuropathology, with possible extreme microlissencephaly with an extremely thin cortex, lissencephaly with either thick or thin/intermediate cortex, and dysgyria combined with cerebellar hypoplasia, pons hypoplasia and corpus callosum dysgenesis. More than 100 MCD-associated mutations have been reported in TUBA1A, TUBB2B, or TUBB3 genes, whereas fewer than ten are known in other genes such TUBB2A, TUBB or TUBG1. Although these mutations are scattered along the α- and ß-tubulin sequences, recurrent mutations are consistently associated with almost identical cortical dysgenesis. Much of the evidence supports that these mutations alter the dynamic properties and functions of microtubules in several fashions. These include diminishing the abundance of functional tubulin heterodimers, altering GTP binding, altering longitudinal and lateral protofilament interactions, and impairing microtubule interactions with kinesin and/or dynein motors or with MAPs. In this review we discuss the recent advances in our understanding of the effects of mutations of tubulins and microtubule-associated proteins on human brain development and the pathogenesis of malformations of cortical development.

Calcium and activity-dependent signaling in the developing cerebral cortex.

Calcium influx can be stimulated by various intra- and extracellular signals to set coordinated gene expression programs into motion. As such, the precise regulation of intracellular calcium represents a nexus between environmental cues and intrinsic genetic programs. Mounting genetic evidence points to a role for the deregulation of intracellular calcium signaling in neuropsychiatric disorders of developmental origin. These findings have prompted renewed enthusiasm for understanding the roles of calcium during normal and dysfunctional prenatal development. In this Review, we describe the fundamental mechanisms through which calcium is spatiotemporally regulated and directs early neurodevelopmental events. We also discuss unanswered questions about intracellular calcium regulation during the emergence of neurodevelopmental disease, and provide evidence that disruption of cell-specific calcium homeostasis and/or redeployment of developmental calcium signaling mechanisms may contribute to adult neurological disorders. We propose that understanding the normal developmental events that build the nervous system will rely on gaining insights into cell type-specific calcium signaling mechanisms. Such an understanding will enable therapeutic strategies targeting calcium-dependent mechanisms to mitigate disease.

Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development.

Corticogenesis consists of a series of synchronised events, including fate transition of cortical progenitors, neuronal migration, specification and connectivity. NeuroD1, a basic helix-loop-helix (bHLH) transcription factor (TF), contributes to all of these events, but how it coordinates these independently is still unknown. Here, we demonstrate that NeuroD1 expression is accompanied by a gain of active chromatin at a large number of genomic loci. Interestingly, transcriptional activation of these loci relied on a high local density of adjacent bHLH TFs motifs, including, predominantly, Tcf12. We found that activity and expression levels of Tcf12 were high in cells with induced levels of NeuroD1 that spanned the transition of cortical progenitors from proliferative to neurogenic divisions. Moreover, Tcf12 forms a complex with NeuroD1 and co-occupies a subset of NeuroD1 target loci. This Tcf12-NeuroD1 cooperativity is essential for gaining active chromatin and targeted expression of genes involved in cell migration. By functional manipulation in vivo, we further show that Tcf12 is essential during cortical development for the correct migration of newborn neurons and, hence, for proper cortical lamination.

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

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