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Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases., Competing Interests: DECLARATION OF INTERESTS J.R.E. serves on the scientific advisory board of Zymo Research Inc.

The neocortex is disproportionately expanded in human compared with mouse 1,2 , both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth 3 . Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer's disease 4,5 . Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease., (© 2021. The Author(s).)

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals 1 . Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations., (© 2021. The Author(s).)

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex., Competing Interests: Declaration of interests L.T.G., T.L.D., J.T.T., E.L., B.K., H.Z., and B.T. are inventors on a PCT application (PCT/US2019/059927) related to this work. All authors declare no other competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Abundant evidence supports the presence of at least three distinct types of thalamocortical (TC) neurons in the primate dorsal lateral geniculate nucleus (dLGN) of the thalamus, the brain region that conveys visual information from the retina to the primary visual cortex (V1). Different types of TC neurons in mice, humans, and macaques have distinct morphologies, distinct connectivity patterns, and convey different aspects of visual information to the cortex. To investigate the molecular underpinnings of these cell types, and how these relate to differences in dLGN between human, macaque, and mice, we profiled gene expression in single nuclei and cells using RNA-sequencing. These efforts identified four distinct types of TC neurons in the primate dLGN: magnocellular (M) neurons, parvocellular (P) neurons, and two types of koniocellular (K) neurons. Despite extensively documented morphological and physiological differences between M and P neurons, we identified few genes with significant differential expression between transcriptomic cell types corresponding to these two neuronal populations. Likewise, the dominant feature of TC neurons of the adult mouse dLGN is high transcriptomic similarity, with an axis of heterogeneity that aligns with core vs. shell portions of mouse dLGN. Together, these data show that transcriptomic differences between principal cell types in the mature mammalian dLGN are subtle relative to the observed differences in morphology and cortical projection targets. Finally, alignment of transcriptome profiles across species highlights expanded diversity of GABAergic neurons in primate versus mouse dLGN and homologous types of TC neurons in primates that are distinct from TC neurons in mouse., Competing Interests: TB, Cv, VM, RH, ZY, TN, LG, GH, DB, JG, AY, EG, SP, TC, SS, EB, AS, BL, ND, KS, SS, AB, JP, MH, CK, GM, EL, HZ, BT No competing interests declared, (© 2021, Bakken et al.)

Single cell/nucleus RNA sequencing (scRNAseq) is emerging as an essential tool to unravel the phenotypic heterogeneity of cells in complex biological systems. While computational methods for scRNAseq cell type clustering have advanced, the ability to integrate datasets to identify common and novel cell types across experiments remains a challenge. Here, we introduce a cluster-to-cluster cell type matching method-FR-Match-that utilizes supervised feature selection for dimensionality reduction and incorporates shared information among cells to determine whether two cell type clusters share the same underlying multivariate gene expression distribution. FR-Match is benchmarked with existing cell-to-cell and cell-to-cluster cell type matching methods using both simulated and real scRNAseq data. FR-Match proved to be a stringent method that produced fewer erroneous matches of distinct cell subtypes and had the unique ability to identify novel cell phenotypes in new datasets. In silico validation demonstrated that the proposed workflow is the only self-contained algorithm that was robust to increasing numbers of true negatives (i.e. non-represented cell types). FR-Match was applied to two human brain scRNAseq datasets sampled from cortical layer 1 and full thickness middle temporal gyrus. When mapping cell types identified in specimens isolated from these overlapping human brain regions, FR-Match precisely recapitulated the laminar characteristics of matched cell type clusters, reflecting their distinct neuroanatomical distributions. An R package and Shiny application are provided at https://github.com/JCVenterInstitute/FRmatch for users to interactively explore and match scRNAseq cell type clusters with complementary visualization tools., (© The Author(s) 2020. Published by Oxford University Press.)

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ("birthdate") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an "inside-out" layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition., Competing Interests: Competing interest statement: T.E.B. and P.R. are coauthors on a 2019 article.

The hippocampal dentate gyrus (DG) is a unique brain region maintaining neural stem cells (NCSs) and neurogenesis into adulthood. We used multiphoton imaging to visualize genetically defined progenitor subpopulations in live slices across key stages of mouse DG development, testing decades old static models of DG formation with molecular identification, genetic-lineage tracing, and mutant analyses. We found novel progenitor migrations, timings, dynamic cell-cell interactions, signaling activities, and routes underlie mosaic DG formation. Intermediate progenitors (IPs, Tbr2+) pioneered migrations, supporting and guiding later emigrating NSCs (Sox9+) through multiple transient zones prior to converging at the nascent outer adult niche in a dynamic settling process, generating all prenatal and postnatal granule neurons in defined spatiotemporal order. IPs (Dll1+) extensively targeted contacts to mitotic NSCs (Notch active), revealing a substrate for cell-cell contact support during migrations, a developmental feature maintained in adults. Mouse DG formation shares conserved features of human neocortical expansion., Competing Interests: BN, RH, RD, PT, SA, KM, RH No competing interests declared, (© 2020, Nelson et al.)

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets. This cluster also shows strong homology to a putative ET cluster in human temporal cortex, but with a strikingly specific regional signature. Together these results suggest that VENs are a regionally distinctive type of ET neuron. Additionally, we describe the first patch clamp recordings of VENs from neurosurgically-resected tissue that show distinctive intrinsic membrane properties relative to neighboring pyramidal neurons.

Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain.

Individuals with sleep apnea often exhibit changes in cognitive behaviors consistent with alterations in the hippocampus. It is hypothesized that adult neurogenesis in the dentate gyrus is an ongoing process that maintains normal hippocampal function in many mammalian species, including humans. However, the impact of chronic intermittent hypoxia (IH), a principal consequence of sleep apnea, on hippocampal adult neurogenesis remains unclear. Using a murine model, we examined the impact of 30 d of IH (IH 30 ) on adult neurogenesis and synaptic plasticity in the dentate gyrus. Although IH 30 did not affect paired-pulse facilitation, IH 30 suppressed long-term potentiation (LTP). Immunohistochemical experiments also indicate that IH perturbs multiple aspects of adult neurogenesis. IH 30 increased the number of proliferating Sox2 + neural progenitor cells in the subgranular zone yet reduced the number of doublecortin-positive neurons. Consistent with these findings, cell lineage tracing revealed that IH 30 increased the proportion of radial glial cells in the subgranular zone, yet decreased the proportion of adult-born neurons in the dentate gyrus. While administration of a superoxide anion scavenger during IH did not prevent neural progenitor cell proliferation, it mitigated the IH-dependent suppression of LTP and prevented adult-born neuron loss. These data demonstrate that IH causes both reactive oxygen species-dependent and reactive oxygen species-independent effects on adult neurogenesis and synaptic plasticity in the dentate gyrus. Our findings identify cellular and neurophysiological changes in the hippocampus that may contribute to cognitive and behavioral deficits occurring in sleep apnea. SIGNIFICANCE STATEMENT Individuals with sleep apnea experience periods of intermittent hypoxia (IH) that can negatively impact many aspects of brain function. Neurons are continually generated throughout adulthood to support hippocampal physiology and behavior. This study demonstrates that IH exposure attenuates hippocampal long-term potentiation and reduces adult neurogenesis. Antioxidant treatment mitigates these effects indicating that oxidative signaling caused by IH is a significant factor that impairs synaptic plasticity and reduces adult neurogenesis in the hippocampus., (Copyright © 2019 the authors 0270-6474/19/391320-12$15.00/0.)

Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues., Competing Interests: The authors have declared that no competing interests exist.

Gene expression studies suggest that differential ion channel expression contributes to differences in rodent versus human neuronal physiology. We tested whether h-channels more prominently contribute to the physiological properties of human compared to mouse supragranular pyramidal neurons. Single-cell/nucleus RNA sequencing revealed ubiquitous HCN1-subunit expression in excitatory neurons in human, but not mouse, supragranular layers. Using patch-clamp recordings, we found stronger h-channel-related membrane properties in supragranular pyramidal neurons in human temporal cortex, compared to mouse supragranular pyramidal neurons in temporal association area. The magnitude of these differences depended upon cortical depth and was largest in pyramidal neurons in deep L3. Additionally, pharmacologically blocking h-channels produced a larger change in membrane properties in human compared to mouse neurons. Finally, using biophysical modeling, we provide evidence that h-channels promote the transfer of theta frequencies from dendrite-to-soma in human L3 pyramidal neurons. Thus, h-channels contribute to between-species differences in a fundamental neuronal property., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Epigenetic factors (EFs) regulate multiple aspects of cerebral cortex development, including proliferation, differentiation, laminar fate, and regional identity. The same neurodevelopmental processes are also regulated by transcription factors (TFs), notably the Pax6→ Tbr2→ Tbr1 cascade expressed sequentially in radial glial progenitors (RGPs), intermediate progenitors, and postmitotic projection neurons, respectively. Here, we studied the EF landscape and its regulation in embryonic mouse neocortex. Microarray and in situ hybridization assays revealed that many EF genes are expressed in specific cortical cell types, such as intermediate progenitors, or in rostrocaudal gradients. Furthermore, many EF genes are directly bound and transcriptionally regulated by Pax6, Tbr2, or Tbr1, as determined by chromatin immunoprecipitation-sequencing and gene expression analysis of TF mutant cortices. Our analysis demonstrated that Pax6, Tbr2, and Tbr1 form a direct feedforward genetic cascade, with direct feedback repression. Results also revealed that each TF regulates multiple EF genes that control DNA methylation, histone marks, chromatin remodeling, and non-coding RNA. For example, Tbr1 activates Rybp and Auts2 to promote the formation of non-canonical Polycomb repressive complex 1 (PRC1). Also, Pax6, Tbr2, and Tbr1 collectively drive massive changes in the subunit isoform composition of BAF chromatin remodeling complexes during differentiation: for example, a novel switch from Bcl7c (Baf40c) to Bcl7a (Baf40a), the latter directly activated by Tbr2. Of 11 subunits predominantly in neuronal BAF, 7 were transcriptionally activated by Pax6, Tbr2, or Tbr1. Using EFs, Pax6→ Tbr2→ Tbr1 effect persistent changes of gene expression in cell lineages, to propagate features such as regional and laminar identity from progenitors to neurons.

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth.

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The mammalian neocortical progenitor cell niche is composed of a diverse repertoire of neuroepithelial cells, radial glia (RG), and intermediate neurogenic progenitors (INPs). Previously, live-cell imaging experiments have proved crucial in identifying these distinct progenitor populations, especially INPs, which amplify neural output by undergoing additional rounds of proliferation before differentiating into new neurons. INPs also provide feedback to the RG pool by serving as a source of Delta-like 1 (Dll1), a key ligand for activating Notch signaling in neighboring cells, a well-known mechanism for maintaining RG identity. While much is known about Dll1-Notch signaling at the molecular level, little is known about how this cell-cell contact dependent feedback is transmitted at the cellular level. To investigate how RG and INPs might interact to convey Notch signals, we used high-resolution live-cell multiphoton microscopy (MPM) to directly observe cellular interactions and dynamics, in conjunction with Notch-pathway specific reporters in the neocortical neural stem cell niche in organotypic brain slices from embryonic mice. We found that INPs and RG interact via dynamic and transient elongate processes, some apparently long-range (extending from the subventricular zone to the ventricular zone), and some short-range (filopodia-like). Gene expression profiling of RG and INPs revealed further progenitor cell diversification, including different subpopulations of Hes1+ and/or Hes5+ RG, and Dll1+ and/or Dll3+ INPs. Thus, the embryonic progenitor niche includes a network of dynamic cell-cell interactions, using different combinations of Notch signaling molecules to maintain and likely diversify progenitor pools.

The cortical area map is initially patterned by transcription factor (TF) gradients in the neocortical primordium, which define a "protomap" in the embryonic ventricular zone (VZ). However, mechanisms that propagate regional identity from VZ progenitors to cortical plate (CP) neurons are unknown. Here we show that the VZ, subventricular zone (SVZ), and CP contain distinct molecular maps of regional identity, reflecting different gene expression gradients in radial glia progenitors, intermediate progenitors, and projection neurons, respectively. The "intermediate map" in the SVZ is modulated by Eomes (also known as Tbr2), a T-box TF. Eomes inactivation caused rostrocaudal shifts in SVZ and CP gene expression, with loss of corticospinal axons and gain of corticotectal projections. These findings suggest that cortical areas and connections are shaped by sequential maps of regional identity, propagated by the Pax6 → Eomes → Tbr1 TF cascade. In humans, PAX6, EOMES, and TBR1 have been linked to intellectual disability and autism.

The dentate gyrus (DG) is a unique cortical region whose protracted development spans the embryonic and early postnatal periods. DG development involves large-scale reorganization of progenitor cell populations, ultimately leading to the establishment of the subgranular zone neurogenic niche. In the developing DG, the T-box transcription factor Tbr2 is expressed in both Cajal-Retzius cells derived from the cortical hem that guide migration of progenitors and neurons to the DG, and intermediate neuronal progenitors born in the dentate neuroepithelium that give rise to granule neurons. Here we show that in mice Tbr2 is required for proper migration of Cajal-Retzius cells to the DG; and, in the absence of Tbr2, formation of the hippocampal fissure is abnormal, leading to aberrant development of the transhilar radial glial scaffold and impaired migration of progenitors and neuroblasts to the developing DG. Furthermore, loss of Tbr2 results in decreased expression of Cxcr4 in migrating cells, leading to a premature burst of granule neurogenesis during early embryonic development accompanied by increased cell death in mutant animals. Formation of the transient subpial neurogenic zone was abnormal in Tbr2 conditional knock-outs, and the stem cell population in the DG was depleted before proper establishment of the subgranular zone. These studies indicate that Tbr2 is explicitly required for morphogenesis of the DG and participates in multiple aspects of the intricate developmental process of this structure.

Neurogenesis, the production of new neurons, occurs in two specialized niches in the adult brain, the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ) adjacent to the lateral ventricles. In the SGZ, neural stem cells (NSCs) give rise to glutamatergic granule neurons that integrate into the granule cell layer. In the SVZ, NSCs generate a more diverse cohort of new neurons, including GABAergic, dopaminergic, and glutamatergic neurons, all of which migrate to the olfactory bulb through the rostral migratory stream. In both adult neurogenic niches, specific transcription factors have been shown to direct fate specification and lineage commitment. This review summarizes current progress on the transcriptional control of glutamatergic neurogenesis in the SGZ and SVZ, highlighting commonalities as well as differences in their transcriptional programs. In particular, we focus on work from our laboratory and others indicating that precise, sequential expression of transcription factors regulates the progression from NSC to lineage-committed progenitor, and ultimately regulates the production and differentiation of adult-born glutamatergic neurons.

Neurogenesis in the dentate gyrus has been implicated in cognitive functions, including learning and memory, and may be abnormal in major neuropsychiatric disorders, such as depression. Dentate neurogenesis is regulated by interactions between extrinsic factors and intrinsic transcriptional cascades that are currently not well understood. Here we show that Tbr2 (also known as Eomes), a T-box transcription factor expressed by intermediate neuronal progenitors (INPs), is critically required for neurogenesis in the dentate gyrus of developing and adult mice. In the absence of Tbr2, INPs are depleted despite augmented neural stem cell (NSC) proliferation, and neurogenesis is halted as the result of failed neuronal differentiation. Interestingly, we find that Tbr2 likely promotes lineage progression from NSC to neuronal-specified INP in part by repression of Sox2, a key determinant of NSC identity. These findings suggest that Tbr2 expression in INPs is critical for neuronal differentiation in the dentate gyrus and that INPs are an essential stage in the lineage from NSCs to new granule neurons in the dentate gyrus.

Traumatic brain injury (TBI) is a major cause of epilepsy, yet the mechanisms underlying the progression from TBI to epilepsy are unknown. TBI induces the expression of COX-2 (cyclooxygenase-2) and increases levels of prostaglandin E2 (PGE2). Here, we demonstrate that acutely applied PGE2 (2 mum) decreases neocortical network activity by postsynaptically reducing excitatory synaptic transmission in acute and organotypic neocortical slices of mice. In contrast, long-term exposure to PGE2 (2 mum; 48 h) presynaptically increases excitatory synaptic transmission, leading to a hyperexcitable network state that is characterized by the generation of paroxysmal depolarization shifts (PDSs). PDSs were also evoked as a result of depriving organotypic slices of activity by treating them with tetrodotoxin (TTX, 1 mum; 48 h). This treatment predominantly increased postsynaptically excitatory synaptic transmission. The network and cellular effects of PGE2 and TTX treatments reversed within 1 week. Differences in the underlying mechanisms (presynaptic vs postsynaptic) as well as occlusion experiments in which slices were exposed to TTX plus PGE2 suggest that the two substances evoke distinct forms of homeostatic plasticity, both of which result in a hyperexcitable network state. PGE2 and TTX (alone or together with PGE2) also increased levels of apoptotic cell death in organotypic slices. Thus, we hypothesize that the increase in excitability and apoptosis may constitute the first steps in a cascade of events that eventually lead to epileptogenesis triggered by TBI.

Areas and layers of the cerebral cortex are specified by genetic programs that are initiated in progenitor cells and then, implemented in postmitotic neurons. Here, we report that Tbr1, a transcription factor expressed in postmitotic projection neurons, exerts positive and negative control over both regional (areal) and laminar identity. Tbr1 null mice exhibited profound defects of frontal cortex and layer 6 differentiation, as indicated by down-regulation of gene-expression markers such as Bcl6 and Cdh9. Conversely, genes that implement caudal cortex and layer 5 identity, such as Bhlhb5 and Fezf2, were up-regulated in Tbr1 mutants. Tbr1 implements frontal identity in part by direct promoter binding and activation of Auts2, a frontal cortex gene implicated in autism. Tbr1 regulates laminar identity in part by downstream activation or maintenance of Sox5, an important transcription factor controlling neuronal migration and corticofugal axon projections. Similar to Sox5 mutants, Tbr1 mutants exhibit ectopic axon projections to the hypothalamus and cerebral peduncle. Together, our findings show that Tbr1 coordinately regulates regional and laminar identity of postmitotic cortical neurons.

Autism susceptibility candidate 2 (Auts2) is a gene associated with autism and mental retardation, whose function is unknown. Expression of Auts2 mRNA and protein were studied in the developing mouse brain by in situ hybridization, immunohistochemistry, and western blotting. Auts2 mRNA was highly expressed in the developing cerebral cortex and cerebellum, regions often affected by neuropathological changes in autism, and a few other brain regions. On embryonic day (E) 12, Auts2 mRNA was expressed in the cortical preplate, where it colocalized with Tbr1, a transcription factor specific for postmitotic projection neurons. From E16 to postnatal day 21, Auts2 was expressed most abundantly in frontal cortex, hippocampus and cerebellum, including Purkinje cells and deep nuclei. High levels of Auts2 were also detected in developing dorsal thalamus, olfactory bulb, inferior colliculus and substantia nigra. Auts2 protein showed similar regional expression patterns as the mRNA. At the cellular level, Auts2 protein was localized in the nuclei of neurons and some neuronal progenitors.

Neurogenesis in the adult hippocampus is a highly regulated process that originates from multipotent progenitors in the subgranular zone (SGZ). Currently, little is known about molecular mechanisms that regulate proliferation and differentiation in the SGZ. To study the role of transcription factors (TFs), we focused on Tbr2 (T-box brain gene 2), which has been implicated previously in developmental glutamatergic neurogenesis. In adult mouse hippocampus, Tbr2 protein and Tbr2-GFP (green fluorescent protein) transgene expression were specifically localized to intermediate-stage progenitor cells (IPCs), a type of transit amplifying cells. The Tbr2+ IPCs were highly responsive to neurogenic stimuli, more than doubling after voluntary wheel running. Notably, the Tbr2+ IPCs formed cellular clusters, the average size of which (Tbr2+ cells per cluster) likewise more than doubled in runners. Conversely, Tbr2+ IPCs were selectively depleted by antimitotic drugs, known to suppress neurogenesis. After cessation of antimitotic treatment, recovery of neurogenesis was paralleled by recovery of Tbr2+ IPCs, including a transient rebound above baseline numbers. Finally, Tbr2 was examined in the context of additional TFs that, together, define a TF cascade in embryonic neocortical neurogenesis (Pax6 --> Ngn2 --> Tbr2 --> NeuroD --> Tbr1). Remarkably, the same TF cascade was found to be linked to stages of neuronal lineage progression in adult SGZ. These results suggest that Tbr2+ IPCs play a major role in the regulation of adult hippocampal neurogenesis, and that a similar transcriptional program controls neurogenesis in adult SGZ as in embryonic cerebral cortex.

Neurogenesis in the developing cerebral cortex of mice occurs in the dorsal telencephalon between embryonic day 11 (E11) and E17, during which time the majority of cortical projection neurons and some glia are produced from proliferating neuroepithelial cells in the ventricular zone. The number of cells produced by this process is governed by several factors, including cell cycle kinetics and the proportion of daughter cells exiting the cell cycle after a given round of cell division. The in vivo effects of IGF-I on cell cycle kinetics were investigated in nestin/IGF-I transgenic (Tg) embryos, in which IGF-I is overexpressed in the cerebral cortex and dorsal telencephalon. These Tg mice have been shown to exhibit increased cell number in the cortical plate by E16 and increased numbers of neurons and glia in the cerebral cortex during postnatal development. Cumulative S phase labeling with 5-bromo-2'-deoxyuridine revealed a decrease in total cell cycle length (TC) in Tg embryos on E14. This decrease in TC was found to result entirely from a reduction in the length of the G1 phase of the cell cycle from 10.66 to 8.81 hr, with no significant changes in the lengths of the S, G2, and M phases. Additionally, the proportion of daughter cells reentering the cell cycle was significantly increased by 15% in Tg embryos on E14-E15 compared with littermate controls. These data demonstrate that IGF-I regulates progenitor cell division in the ventricular zone by reducing G1 phase length and decreasing TC but increases cell cycle reentry.