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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., Little is known about von Economo neurons, which have been described in a subset of mammals and appear to be selectively lost in several human neurological diseases. Here, authors reveal the gene expression profile of these cells and show that they are likely long-distance projection neurons.

Increasing interest in studies of prenatal human brain development, particularly using new single-cell genomics and anatomical technologies to create cell atlases, creates a strong need for accurate and detailed anatomical reference atlases. In this study, we present two cellular-resolution digital anatomical atlases for prenatal human brain at post-conceptional weeks (PCW) 15 and 21. Both atlases were annotated on sequential Nissl-stained sections covering brain-wide structures on the basis of combined analysis of cytoarchitecture, acetylcholinesterase staining and an extensive marker gene expression dataset. This high information content dataset allowed reliable and accurate demarcation of developing cortical and subcortical structures and their subdivisions. Furthermore, using the anatomical atlases as a guide, spatial expression of 37 and 5 genes from the brains respectively at PCW 15 and 21 was annotated, illustrating reliable marker genes for many developing brain structures. Finally, the present study uncovered several novel developmental features, such as the lack of an outer subventricular zone in the hippocampal formation and entorhinal cortex, and the apparent extension of both cortical (excitatory) and subcortical (inhibitory) progenitors into the prenatal olfactory bulb. These comprehensive atlases provide useful tools for visualization, segmentation, targeting, imaging and interpretation of brain structures of prenatal human brain, and for guiding and interpreting the next generation of cell census and connectome studies. This article is protected by copyright. All rights reserved.

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To systematically profile morpho-electric properties of mammalian neurons, we established a single-cell characterization pipeline using standardized patch-clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly accessible online database, the Allen Cell Types Database, to display these datasets. Intrinsic physiological properties were measured from 1,938 neurons from the adult laboratory mouse visual cortex, morphological properties were measured from 461 reconstructed neurons, and 452 neurons had both measurements available. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We established a taxonomy of morphologically and electrophysiologically defined cell types for this region of the cortex, with 17 electrophysiological types, 38 morphological types and 46 morpho-electric types. There was good correspondence with previously defined transcriptomic cell types and subclasses using the same transgenic mouse lines.

SUMMARY Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-sub-class-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools., Graphical Abstract, In brief Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Mich et al. use comparative open chromatin analysis to identify human neocortical enhancers that can drive gene expression from AAV vectors in a subclass-specific fashion in multiple species.

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.

Background The complex interspersed pattern of segmental duplications in humans is responsible for rearrangements associated with neurodevelopmental disease, including the emergence of novel genes important in human brain evolution. We investigate the evolution of LCR16a, a putative driver of this phenomenon that encodes one of the most rapidly evolving human–ape gene families, nuclear pore interacting protein (NPIP). Results Comparative analysis shows that LCR16a has independently expanded in five primate lineages over the last 35 million years of primate evolution. The expansions are associated with independent lineage-specific segmental duplications flanking LCR16a leading to the emergence of large interspersed duplication blocks at non-orthologous chromosomal locations in each primate lineage. The intron-exon structure of the NPIP gene family has changed dramatically throughout primate evolution with different branches showing characteristic gene models yet maintaining an open reading frame. In the African ape lineage, we detect signatures of positive selection that occurred after a transition to more ubiquitous expression among great ape tissues when compared to Old World and New World monkeys. Mouse transgenic experiments from baboon and human genomic loci confirm these expression differences and suggest that the broader ape expression pattern arose due to mutational changes that emerged in cis. Conclusions LCR16a promotes serial interspersed duplications and creates hotspots of genomic instability that appear to be an ancient property of primate genomes. Dramatic changes to NPIP gene structure and altered tissue expression preceded major bouts of positive selection in the African ape lineage, suggestive of a gene undergoing strong adaptive evolution.

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled ∼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.

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.

Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties.

Recent large-scale international collaborations are generating major surveys of cell types and connections in the mouse brain, collecting large amounts of data across modalities, spatial scales, and brain areas. Successful integration of these data requires a standard 3D reference atlas. Here, we present the Allen Mouse Brain Common Coordinate Framework (CCFv3) as such a resource. We constructed an average template brain at 10 μm voxel resolution by interpolating high resolution in-plane serial two-photon tomography images with 100 μm z-sampling from 1,675 young adult C57BL/6J mice. Then, using multimodal reference data, we parcellated the entire brain directly in 3D, labeling every voxel with a brain structure spanning 43 isocortical areas and their layers, 329 subcortical gray matter structures, 81 fiber tracts, and 8 ventricular structures. CCFv3 can be used to analyze, visualize and integrate multimodal and multiscale datasets in 3D, and is openly accessible (https://atlas.brain-map.org).

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.

Developmental changes in the temporal and spatial regulation of gene expression drive the emergence of normal mature brain function, while disruptions in these processes underlie many neurodevelopmental abnormalities. To solidify our foundational knowledge of such changes in a primate brain with an extended period of postnatal maturation like in human, we investigated the whole-genome transcriptional profiles of rhesus monkey brains from birth to adulthood. We found that gene expression dynamics are largest from birth through infancy, after which gene expression profiles transition to a relatively stable state by young adulthood. Biological pathway enrichment analysis revealed that genes more highly expressed at birth are associated with cell adhesion and neuron differentiation, while genes more highly expressed in juveniles and adults are associated with cell death. Neocortex showed significantly greater differential expression over time than subcortical structures, and this trend likely reflects the protracted postnatal development of the cortex. Using network analysis, we identified 27 co-expression modules containing genes with highly correlated expression patterns that are associated with specific brain regions, ages or both. In particular, one module with high expression in neonatal cortex and striatum that decreases during infancy and juvenile development was significantly enriched for autism spectrum disorder (ASD)-related genes. This network was enriched for genes associated with axon guidance and interneuron differentiation, consistent with a disruption in the formation of functional cortical circuitry in ASD.

The Allen Mouse Brain Connectivity Atlas is a mesoscale whole brain axonal projection atlas of the C57Bl/6J mouse brain. Anatomical trajectories throughout the brain were mapped into a common 3D space using a standardized platform to generate a comprehensive and quantitative database of inter-areal and cell-type-specific projections. This connectivity atlas has several desirable features, including brain-wide coverage, validated and versatile experimental techniques, a single standardized data format, a quantifiable and integrated neuroinformatics resource, and an open-access public online database (http://connectivity.brain-map.org/). Meaningful informatics data quantification and comparison is key to effective use and interpretation of connectome data. This relies on successful definition of a high fidelity atlas template and framework, mapping precision of raw data sets into the 3D reference framework, accurate signal detection and quantitative connection strength algorithms, and effective presentation in an integrated online application. Here we describe key informatics pipeline steps in the creation of the Allen Mouse Brain Connectivity Atlas and include basic application use cases.

The neurogenic potential of the subgranular zone (SGZ) of the hippocampal dentate gyrus is likely to be regulated by molecular cues arising from its complex heterogeneous cellular environment. Through transcriptome analysis using laser microdissection coupled with DNA microarrays, in combination with analysis of genome-wide in situ hybridization data, we identified 363 genes selectively enriched in adult mouse SGZ. These genes reflect expression in the different constituent cell types, including progenitor and dividing cells, immature granule cells, astrocytes, oligodendrocytes and GABAergic interneurons. Similar transcriptional profiling in the rhesus monkey dentate gyrus across postnatal development identified a highly overlapping set of SGZ-enriched genes, which can be divided based on temporal profiles to reflect maturation of glia versus granule neurons. Furthermore, we identified a neurogenesis-related gene network with decreasing postnatal expression that is highly correlated with the declining number of proliferating cells in dentate gyrus over postnatal development. Many of the genes in this network showed similar postnatal downregulation in mouse, suggesting a conservation of molecular mechanisms underlying developmental and adult neurogenesis in rodents and primates. Conditional deletion of Sox4 and Sox11, encoding two neurogenesis-related transcription factors central in this network, produces a mouse with no hippocampus, confirming the crucial role for these genes in regulating hippocampal neurogenesis.

The transcriptional underpinnings of brain development remain poorly understood, particularly in humans and closely related non-human primates. We describe a high-resolution transcriptional atlas of rhesus monkey (Macaca mulatta) brain development that combines dense temporal sampling of prenatal and postnatal periods with fine anatomical division of cortical and subcortical regions associated with human neuropsychiatric disease. Gene expression changes more rapidly before birth, both in progenitor cells and maturing neurons. Cortical layers and areas acquire adult-like molecular profiles surprisingly late in postnatal development. Disparate cell populations exhibit distinct developmental timing of gene expression, but also unexpected synchrony of processes underlying neural circuit construction including cell projection and adhesion. Candidate risk genes for neurodevelopmental disorders including primary microcephaly, autism spectrum disorder, intellectual disability, and schizophrenia show disease-specific spatiotemporal enrichment within developing neocortex. Human developmental expression trajectories are more similar to monkey than rodent, although approximately 9% of genes show human-specific regulation with evidence for prolonged maturation or neoteny compared to monkey.

The connections between the claustrum and the cortex in mouse are systematically investigated with adeno‐associated virus (AAV), an anterograde viral tracer. We first define the boundary and the three‐dimensional structure of the claustrum based on a variety of molecular and anatomical data. From AAV injections into 42 neocortical and allocortical areas, we conclude that most cortical areas send bilateral projections to the claustrum, the majority being denser on the ipsilateral side. This includes prelimbic, infralimbic, medial, ventrolateral and lateral orbital, ventral retrosplenial, dorsal and posterior agranular insular, visceral, temporal association, dorsal and ventral auditory, ectorhinal, perirhinal, lateral entorhinal, and anteromedial, posteromedial, lateroposterior, laterointermediate, and postrhinal visual areas. In contrast, the cingulate and the secondary motor areas send denser projections to the contralateral claustrum than to the ipsilateral one. The gustatory, primary auditory, primary visual, rostrolateral visual, and medial entorhinal cortices send projections only to the ipsilateral claustrum. Primary motor, primary somatosensory and subicular areas barely send projections to either ipsi‐ or contralateral claustrum. Corticoclaustral projections are organized in a rough topographic manner, with variable projection strengths. We find that the claustrum, in turn, sends widespread projections preferentially to ipsilateral cortical areas with different projection strengths and laminar distribution patterns and to certain contralateral cortical areas. Our quantitative results show that the claustrum has strong reciprocal and bilateral connections with prefrontal and cingulate areas as well as strong reciprocal connections with the ipsilateral temporal and retrohippocampal areas, suggesting that it may play a crucial role in a variety of cognitive processes. J. Comp. Neurol. 525:1317–1346, 2017. © 2016 Wiley Periodicals, Inc.

The Allen Brain Atlas (http://www.brain-map.org) provides a unique online public resource integrating extensive gene expression data, connectivity data and neuroanatomical information with powerful search and viewing tools for the adult and developing brain in mouse, human and non-human primate. Here, we review the resources available at the Allen Brain Atlas, describing each product and data type [such as in situ hybridization (ISH) and supporting histology, microarray, RNA sequencing, reference atlases, projection mapping and magnetic resonance imaging]. In addition, standardized and unique features in the web applications are described that enable users to search and mine the various data sets. Features include both simple and sophisticated methods for gene searches, colorimetric and fluorescent ISH image viewers, graphical displays of ISH, microarray and RNA sequencing data, Brain Explorer software for 3D navigation of anatomy and gene expression, and an interactive reference atlas viewer. In addition, cross data set searches enable users to query multiple Allen Brain Atlas data sets simultaneously. All of the Allen Brain Atlas resources can be accessed through the Allen Brain Atlas data portal.

Neuroanatomically precise, genome-wide maps of transcript distributions are critical resources to complement genomic sequence data and to correlate functional and genetic brain architecture. Here we describe the generation and analysis of a transcriptional atlas of the adult human brain, comprising extensive histological analysis and comprehensive microarray profiling of ~900 neuroanatomically precise subdivisions in two individuals. Transcriptional regulation varies enormously by anatomical location, with different regions and their constituent cell types displaying robust molecular signatures that are highly conserved between individuals. Analysis of differential gene expression and gene co-expression relationships demonstrates that brain-wide variation strongly reflects the distributions of major cell classes such as neurons, oligodendrocytes, astrocytes and microglia. Local neighbourhood relationships between fine anatomical subdivisions are associated with discrete neuronal subtypes and genes involved with synaptic transmission. The neocortex displays a relatively homogeneous transcriptional pattern, but with distinct features associated selectively with primary sensorimotor cortices and with enriched frontal lobe expression. Notably, the spatial topography of the neocortex is strongly reflected in its molecular topography— the closer two cortical regions, the more similar their transcriptomes. This freely accessible online data resource forms a high-resolution transcriptional baseline for neurogenetic studies of normal and abnormal human brain function.

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

Although cytoarchitectonic organization of the mammalian cortex into different lamina has been well-studied, identifying the architectural differences that distinguish cortical areas from one another is more challenging. Localization of large anatomical structures is possible using magnetic resonance imaging or invasive techniques (such as anterograde or retrograde tracing), but identifying patterns in gene expression architecture is limited as gene products do not necessarily identify an immediate functional consequence of a specialized area. Expression of specific genes in the mouse and human cortex is most often identified across entire lamina, and areal patterning of expression (when it exists) is most easily differentiated on a layer-by-layer basis. Since cortical organization is defined by the expression of large sets of genes, the task of identifying individual (or groups of structures) cannot be done using individual areal markers. In this manuscript we describe a methodology for clustering gene expression correlation profiles in the C57Bl/6J mouse cortex to identify large-scale genetic relationships between layers and areas. By using the Anatomic Gene Expression Atlas (http://mouse.brain-map.org/agea/) derived from in situ hybridization data in the Allen Brain Atlas, we show that a consistent expression based organization of areal patterning in the mouse cortex exists when clustered on a laminar basis. Surface-based mapping and visualization techniques are used as a representation to clarify these relationships.

The Allen Brain Atlas (ABA, www.brain-map.org) is a genome wide, spatially registered collection of cellular resolution in situ hybridization gene expression image data of the C57Bl/6J mouse brain. Derived from the ABA, the Anatomic Gene Expression Atlas (AGEA, http://mouse.brain-map.org/agea) has demonstrated both laminar and areal spatial gene expression correlations in the mouse cortex. While the mouse cortex is lissencephalic, its curvature and substantial bending in boundary areas renders it difficult to visualize and analyze laminar versus areal effects in a rectilinear coordinate framework. In context of human and non-human primate cortex, surface-based representation has proven useful for understanding relative locations of laminar, columnar, and areal features. In this paper, we describe a methodology for constructing surface-based flatmaps of the mouse cortex that enables mapping of gene expression data from individual genes in the ABA, or probabilistic expression maps from the AGEA, to identify and visualize genetic relationships between layers and areas.

The Cre/lox system is widely used in mice to achieve cell-type-specific gene expression. However, a strong and universal responding system to express genes under Cre control is still lacking. We have generated a set of Cre reporter mice with strong, ubiquitous expression of fluorescent proteins of different spectra. The robust native fluorescence of these reporters enables direct visualization of fine dendritic structures and axonal projections of the labeled neurons, which is useful in mapping neuronal circuitry, imaging and tracking specific cell populations in vivo. Using these reporters and a high-throughput in situ hybridization platform, we are systematically profiling Cre-directed gene expression throughout the mouse brain in a number of Cre-driver lines, including novel Cre lines targeting different cell types in the cortex. Our expression data are displayed in a public online database to help researchers assess the utility of various Cre-driver lines for cell-type-specific genetic manipulation.

The Sox2 gene is a key regulator of pluripotency embedded within an intron of a long noncoding RNA (ncRNA), termed Sox2 overlapping transcript (Sox2ot), which is transcribed in the same orientation. However, this ncRNA remains uncharacterized. Here we show that Sox2ot has multiple transcription start sites associated with genomic features that indicate regulated expression, including highly conserved elements (HCEs) and chromatin marks characteristic of gene promoters. To identify biological processes in which Sox2ot may be involved, we analyzed its expression in several developmental systems, compared to expression of Sox2. We show that Sox2ot is a stable transcript expressed in mouse embryonic stem cells, which, like Sox2, is down-regulated upon induction of embryoid body (EB) differentiation. However, in contrast to Sox2, Sox2ot is up-regulated during EB mesoderm-lineage differentiation. In adult mouse, Sox2ot isoforms were detected in tissues where Sox2 is expressed, as well as in different tissues, supporting independent regulation of expression of the ncRNA. Sox2dot, an isoform of Sox2ot transcribed from a distal HCE located >500 kb upstream of Sox2, was detected exclusively in the mouse brain, with enrichment in regions of adult neurogenesis. In addition, Sox2ot isoforms are transcribed from HCEs upstream of Sox2 in other vertebrates, including in several regions of the human brain. We also show that Sox2ot is dynamically regulated during chicken and zebrafish embryogenesis, consistently associated with central nervous system structures. These observations provide insight into the structure and regulation of the Sox2ot gene, and suggest conserved roles for Sox2ot orthologs during vertebrate development.

Molecular approaches to understanding the functional circuitry of the nervous system promise new insights into the relationship between genes, brain and behaviour. The cellular diversity of the brain necessitates a cellular resolution approach towards understanding the functional genomics of the nervous system. We describe here an anatomically comprehensive digital atlas containing the expression patterns of approximately 20,000 genes in the adult mouse brain. Data were generated using automated high-throughput procedures for in situ hybridization and data acquisition, and are publicly accessible online. Newly developed image-based informatics tools allow global genome-scale structural analysis and cross-correlation, as well as identification of regionally enriched genes. Unbiased fine-resolution analysis has identified highly specific cellular markers as well as extensive evidence of cellular heterogeneity not evident in classical neuroanatomical atlases. This highly standardized atlas provides an open, primary data resource for a wide variety of further studies concerning brain organization and function.

Several large-scale projects are evaluating gene expression in the mouse brain, both spatially and temporally. These range from projects that cover a broad spectrum of genes and developmental stages to those with high-spatial resolution and gene coverage but for only a single developmental stage. Each project contains its own self-consistent data set and tools for analysis and mining. Preliminary efforts are under way to construct tools and an infrastructure with which the data from across these different projects can be statistically pooled and analyzed. However, many obstacles remain, and these must be addressed and overcome soon if we are to unify the data sets, otherwise the preliminary efforts will be wasted. Here, the various projects for collecting and mining this information are reviewed, some challenges in data set comparisons are discussed, and some basic proposals are made for overcoming the challenges.

The Allen Mouse Brain Connectivity Atlas is a mesoscale whole brain axonal projection atlas of the C57Bl/6J mouse brain. All data were aligned to a common template in 3D space to generate a comprehensive and quantitative database of inter-areal and cell-type-specific projections. A suite of computational tools were developed to search and visualize the projection labeling experiments, available at http://connectivity.brain-map.org. We present three use cases illustrating how these publicly-available tools can be used to perform analyses of long range brain region connectivity. The use cases make extensive use of advanced visualization tools integrated with the atlas including projection density histograms, 3D computed anterograde and retrograde projection paths, and multi-specimen projection composites. These tools offer convenient access to detailed axonal projection information in the adult mouse brain and the ability to perform data analysis and visualization of projection fields and neuroanatomy in an integrated manner.

Significant advances in circuit-level analyses of the brain require tools that allow for labeling, modulation of gene expression, and monitoring and manipulation of cellular activity in specific cell types and/or anatomical regions. Large-scale projects and individual laboratories have produced hundreds of gene-specific promoter-driven Cre mouse lines invaluable for enabling genetic access to subpopulations of cells in the brain. However, the potential utility of each line may not be fully realized without systematic whole brain characterization of transgene expression patterns. We established a high-throughput in situ hybridization (ISH), imaging and data processing pipeline to describe whole brain gene expression patterns in Cre driver mice. Currently, anatomical data from over 100 Cre driver lines are publicly available via the Allen Institute's Transgenic Characterization database, which can be used to assist researchers in choosing the appropriate Cre drivers for functional, molecular, or connectional studies of different regions and/or cell types in the brain.

The anatomical and functional architecture of the human brain is mainly determined by prenatal transcriptional processes. We describe an anatomically comprehensive atlas of the mid-gestational human brain, including de novo reference atlases, in situ hybridization, ultra-high-resolution magnetic resonance imaging (MRI) and microarray analysis on highly discrete laser-microdissected brain regions. In developing cerebral cortex, transcriptional differences are found between different proliferative and post-mitotic layers, wherein laminar signatures reflect cellular composition and developmental processes. Cytoarchitectural differences between human and mouse have molecular correlates, including species differences in gene expression in subplate, although surprisingly we find minimal differences between the inner and outer subventricular zones even though the outer zone is expanded in humans. Both germinal and post-mitotic cortical layers exhibit fronto-temporal gradients, with particular enrichment in the frontal lobe. Finally, many neurodevelopmental disorder and human-evolution-related genes show patterned expression, potentially underlying unique features of human cortical formation. These data provide a rich, freely-accessible resource for understanding human brain development.

Leishmania are evolutionarily ancient protozoans (Kinetoplastidae) and important human pathogens that cause a spectrum of diseases ranging from the asymptomatic to the lethal. The Leishmania genome is relatively small [≈34 megabases (Mb)], lacks substantial repetitive DNA, and is distributed among 36 chromosomes pairs ranging in size from 0.3 Mb to 2.5 Mb, making it a useful candidate for complete genome sequence determination. We report here the nucleotide sequence of the smallest chromosome, chr1. The sequence of chr1 has a 257-kilobase region that is densely packed with 79 protein-coding genes. This region is flanked by telomeric and subtelomeric repetitive elements that vary in number and content among the chr1 homologs, resulting in an ≈27.5-kilobase size difference. Strikingly, the first 29 genes are all encoded on one DNA strand, whereas the remaining 50 genes are encoded on the opposite strand. Based on the gene density of chr1, we predict a total of ≈9,800 genes in Leishmania , of which 40% may encode unknown proteins.

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii consists of a heterogeneous family of proteins that are encoded by approximately 100 unique genes. A genomic expression library was screened with a panel of MSG-specific monoclonal antibodies (MAbs) to identify conserved and rare epitopes. All of the antibodies reacted with epitopes that are encoded within the 5′ end of MSG. The results from the expression screening identified antibodies that recognize highly conserved, moderately conserved, and rare epitopes. Four MAbs (MAbs RA-F1, RA-E7, RA-G10, and RB-E3) reacted with a maltose binding protein–MSG-B fusion protein ( MBP MSG-B 41–1065 ) by immunoblotting and enzyme-linked immunosorbent assay. Three of the MAbs (MAbs RA-F1, RA-G10, and RA-E7) reacted with the same continuous epitope that was localized to amino acids 278 to 290 of MSG-B. Comparison of the sequence of the RA-F1-, RA-G10-, and RA-E7-reactive epitope to the deduced amino acid sequences of multiple MSGs demonstrated that it is highly conserved. The reactivity of RB-E3 with MSG-B was shown to be dependent on amino acids 184 to 192, which may comprise a portion of a discontinuous epitope.

Large-scale databases of the brain are providing content to the neuroscience community through molecular, cellular, functional, and connectomic data. Organization, presentation, and maintenance requirements are substantial given the complexity, diverse modalities, resolution, and scale. In addition to microarrays, magnetic resonance imaging, and RNA sequencing, several in situ hybridization databases have been constructed due to their value in spatially localizing cellular expression. Scalable techniques for processing and presenting these data for maximum utility in viewing and analysis are key for end user value. We describe methods and use cases for the Allen Brain Atlas resources of the adult and developing mouse.

Genes encoding the TFIID TATA-box binding protein (TBP) from two probable species of rat Pneumocystis carinii (prototype and variant) were sequenced. The two P. carinii TBP gene sequences were 91% identical to each other, and 65-77% identical to TBP genes from other species. A cDNA from one of the two P. carinii TBP genes was sequenced, which showed that four small introns resided in identical positions within the TBP genes from the prototype and variant rat P. carinii. Conservation of the 180 amino acids that constitute the conserved core of TBP was 97% between the P. carinii TBP, which were 95% and 97% identical to conserved core sequences of TBP from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively.

A fragment from the genome of rat-derived Pneumocystis carinii was found to contain two MSG genes arranged as a direct repeat. The sequences from one gene (MSG B), the region between the two genes, and part of the second gene (MSG A) were determined. The two MSG genes were not identical in sequence. The open reading frames of MSG A and MSG B encode non-identical proteins, both of which are similar to that encoded by a previously published cDNA. The MSG B gene sequence showed no evidence of introns. The 5’and 3’untranslated regions of the MSG gene pair were highly conserved, but the regions immediately upstream of the open reading frames of MSG A and B were different from the region upstream of a previously characterized MSG cDNA. Primers designed to extend upstream of the 5’end of MSG and downstream of the 3’end of MSG were used in a polymerase chain reaction with total genomic P. carinii DNA as template. Presumptive intergenic amplification products from this reaction were cloned and sequenced. The sequences of these regions were similar but distinct, indicating that tandem arrangement of MSG genes is a common organizational motif.

SummarySpinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder caused by CAG/polyglutamine repeat expansions in the ataxin-7 gene. Ataxin-7 is a component of two different transcription coactivator complexes, and recent work indicates that disease protein normal function is altered in polyglutamine neurodegeneration. Given this, we studied how ataxin-7 gene expression is regulated. The ataxin-7 repeat and translation start site are flanked by binding sites for CTCF, a highly conserved multifunctional transcription regulator. When we analyzed this region, we discovered an adjacent alternative promoter and a convergently transcribed antisense noncoding RNA, SCAANT1. To understand how CTCF regulates ataxin-7 gene expression, we introduced ataxin-7 mini-genes into mice, and found that CTCF is required for SCAANT1 expression. Loss of SCAANT1 derepressed ataxin-7 sense transcription in a cis-dependent fashion and was accompanied by chromatin remodeling. Discovery of this pathway underscores the importance of altered epigenetic regulation for disease pathology at repeat loci exhibiting bidirectional transcription.Video Abstract

The Allen Brain Atlas, a Web-based, genome-wide atlas of gene expression in the adult mouse brain, was an experiment on a massive scale. The development of the atlas faced a combination of great technical challenges and a non-traditional open research model, and it encountered many hurdles on the path to completion and community adoption. Having overcome these challenges, it is now a fundamental tool for neuroscientists worldwide and has set the stage for the creation of other similar open resources. Nevertheless, there are many untapped opportunities for exploration.

The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.

A major proportion of the mammalian transcriptome comprises long RNAs that have little or no protein-coding capacity (ncRNAs). Only a handful of such transcripts have been examined in detail, and it is unknown whether this class of transcript is generally functional or merely artifact. Using in situ hybridization data from the Allen Brain Atlas, we identified 849 ncRNAs (of 1,328 examined) that are expressed in the adult mouse brain and found that the majority were associated with specific neuroanatomical regions, cell types, or subcellular compartments. Examination of their genomic context revealed that the ncRNAs were expressed from diverse places including intergenic, intronic, and imprinted loci and that many overlap with, or are transcribed antisense to, protein-coding genes of neurological importance. Comparisons between the expression profiles of ncRNAs and their associated protein-coding genes revealed complex relationships that, in combination with the specific expression profiles exhibited at both regional and subcellular levels, are inconsistent with the notion that they are transcriptional noise or artifacts of chromatin remodeling. Our results show that the majority of ncRNAs are expressed in the brain and provide strong evidence that the majority of processed transcripts with no protein-coding capacity function intrinsically as RNAs.

The growing number of publicly available databases of murine gene expression arising from genomic-scale transcriptome/proteome profiling projects allows open access to information about genes potentially involved in diseases and disorders of the brain. The use of various methodologies by myriad projects provides complementary types of information, ranging from easily quantifiable microarray data for gross brain regions, to transcript tag analysis and proteomic characterization. One mode of gene expression analysis that has recently been widely adopted is the utilization of colorimetric in situ hybridization. This approach is adaptable for high throughput production, and provides a reproducible, scaleable platform for large datasets. The Allen Brain Atlas in particular has utilized this technology to produce a genomic-scale anatomical digital atlas of gene expression in the adult male mouse brain. The availability of global datasets with cellular level spatial resolution, which can be easily parsed due to accessible informatics-derived image analysis tools, can provide both high level and detailed insights into gene regulation. This article reviews various gene expression profiling projects in the mouse brain, how these data sets are increasingly used to complement other studies and applications of these datasets to further understanding of neurological disease.

We report here a second-generation tetracycline-responsive repressor-operator system in Leishmania donovani. In this system, expression of a reporter luciferase gene (LUC) is driven by the inducible Leishmania ribosomal RNA promoter on the DNA strand opposite to a hygromycin resistance gene (HYG) whose expression is driven by the endogenous pol I promoter on chromosome 27 (rDNA locus) or the endogenous pol II promoter on chromosome 35 (LD1 locus). Transgenic cell lines showed regulation of LUC gene expression over three orders of magnitude. In the absence of tetracycline, luciferase expression levels were 2-3-fold higher than machine background when integrated into the LD1 locus, but was over 10-fold higher than machine background when integrated into the rDNA locus.

The sequencing of Leishmania major Friedlin chromosome 1 (Chr1), Chr3, and Chr4 has been completed. and several other chromosomes are well underway. The complete genome sequence should be available by 2003. Over 1,000 full-length new genes have been identified, with the majority (approximately 75%) having unknown function. Many of these may be Leishmania (or kinetoplastid) specific. Most interestingly, the genes are organized into large (> 100-500 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a "divergent" manner, i.e., the mRNAs for the two sets of genes are both transcribed towards the telomeres. Nuclear run-on analysis suggests that transcription is initiated in both directions within the "divergent" region. Chr3 and Chr4 contain two "convergent" clusters, with a single "divergent" gene at one telomere of Chr3. Sequence analysis of several genes from the LD1 region of Chr35 indicates a high degree of sequence conservation between L. major and L. donovani/L. infantum within protein-coding open reading frames (ORFs), with a lower degree of conservation within the non-coding regions. Immunization of mice with recombinant antigen from two of these genes, BTI (formerly ORFG) and ORFF, results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

The sequence and gene organization of the ribosomal RNA (rRNA) genes of Leishmania major Friedlin (LmjF) were determined. Interestingly, the rDNA repeat unit contained a duplicated 526 bp fragment at the 3' end of the unit with two copies of the LSUepsilon rRNA gene. Our results suggested the presence of only approximately 24 copies of the rRNA unit per diploid genome in LmjF. Repetitive elements (IGSRE) of 63 bp occurred in the intergenic spacer (IGS) between the LSUepsilon and the SSU rRNA genes. Among the different rDNA units, the region containing the IGSRE fluctuated in length from approximately 1.3 to approximately 18 kb. The transcription initiation site (TIS) of the rRNA unit was localized by primer extension to 1043 bp upstream of the SSU gene and 184 bp downstream of the IGSRE. Sequence comparison among several species of Leishmania showed a high degree of conservation around the TIS. Moreover, the IGSRE also showed considerable similarity between Leishmania species. In transient transfection assays, a fragment containing the TIS directed a 164- to 178-fold increase in luciferase activity over the no-insert control, indicating the presence of a promoter within this 391 bp fragment. The LmjF promoter region was also functional in other species of Leishmania. Nuclear run-on analyses demonstrated that only the rRNA-coding strand is transcribed, downstream of this RNA polymerase I (pol I) promoter. These experiments also suggested that transcription terminates upstream of the IGSRE.

a Seattle Biomedical Research Institute, 4 Nickerson Street, Seattle, WA 98109-1651, USA b Rosetta Inpharmatics, 115th A6e NE, Kirkland, WA 98034, USA c Department of Molecular Microbiology, 760 McDonnell Science Building Box 8230, Washington Uni6ersity School of Medicine, 660 Euclid A6enue, St. Louis, MO 63110-1093, USA d Department of Pathobiology, Uni6ersity of Washington, Seattle, WA 98195, USA

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSGs each begin with the same 365-bp sequence, called the Upstream Conserved Sequence (UCS), which is in frame with the contiguous MSG sequence. The UCS contains several potential start sites for translation. To determine if translation of MSG mRNAs begins in the UCS, polyclonal antiserum was raised against the 123-amino-acid peptide encoded by the UCS. The anti-UCS serum reacted with a P. carinii protein that migrated at 170 kDa; however, it did not react with the mature MSG protein, which migrates at 116 kDa. A 170-kDa protein was immunoprecipitated with anti-UCS serum and shown to react with a monoclonal antibody against a conserved MSG epitope. To explore the functional role of the UCS in the trafficking of MSG, the nucleotide sequence encoding the UCS peptide was ligated to the 5′ end of an MSG gene and incorporated into a recombinant baculovirus. Insect cells infected with the UCS-MSG hybrid gene expressed a 160-kDa protein which was N-glycosylated. By contrast, insect cells infected with a baculovirus carrying an MSG gene lacking the UCS expressed a nonglycosylated 130-kDa protein. These data suggest that in P. carinii , translation begins in the UCS to produce a pre-MSG protein, which is subsequently directed to the endoplasmic reticulum and processed to the mature form by proteolytic cleavage.

Pneumocystis is a eukaryotic microbe that causes pneumocystosis, an AIDS-associated pneumonia. Pneumocystosis also occurs in many other mammalian species, and animal-derived organisms have been extensively utilized in Pneumocystis research. Pneumocystis from diverse hosts contain a large glycoprotein (gpA/MSG) on the surface. Antibodies elicited against gpA/MSG of Pneumocystis from humans sometimes cross-react with epitopes on proteins of similar size from Pneumocystis from other host species. Here we report the isolation and partial sequence of two presumptive gpA/MSG genes from human-derived Pneumocystis. The cloned human-derived Pneumocystis gpA/MSG genes and predicted peptides were different from those previously isolated from Pneumocystis from rats and ferrets. The genome of human-derived Pneumocystis contained multiple copies of sequences related to the two cloned gpA/MSG genes.

SummaryAvailability of genome-scale in situ hybridization data allows systematic analysis of genetic neuroanatomical architecture. Within the hippocampus, electrophysiology and lesion and imaging studies demonstrate functional heterogeneity along the septotemporal axis, although precise underlying circuitry and molecular substrates remain uncharacterized. Application of unbiased statistical component analyses to genome-scale hippocampal gene expression data revealed robust septotemporal molecular heterogeneity, leading to the identification of a large cohort of genes with robust regionalized hippocampal expression. Manual mapping of heterogeneous CA3 pyramidal neuron expression patterns demonstrates an unexpectedly complex molecular parcellation into a relatively coherent set of nine expression domains in the septal/temporal and proximal/distal axes with reciprocal, nonoverlapping boundaries. Unique combinatorial profiles of adhesion molecules within these domains suggest corresponding differential connectivity, which is demonstrated for CA3 projections to the lateral septum using retrograde labeling. This complex, discrete molecular architecture provides a novel paradigm for predicting functional differentiation across the full septotemporal extent of the hippocampus.

SummaryGenome-wide transcriptional profiling was used to characterize the molecular underpinnings of neocortical organization in rhesus macaque, including cortical areal specialization and laminar cell-type diversity. Microarray analysis of individual cortical layers across sensorimotor and association cortices identified robust and specific molecular signatures for individual cortical layers and areas, prominently involving genes associated with specialized neuronal function. Overall, transcriptome-based relationships were related to spatial proximity, being strongest between neighboring cortical areas and between proximal layers. Primary visual cortex (V1) displayed the most distinctive gene expression compared to other cortical regions in rhesus and human, both in the specialized layer 4 as well as other layers. Laminar patterns were more similar between macaque and human compared to mouse, as was the unique V1 profile that was not observed in mouse. These data provide a unique resource detailing neocortical transcription patterns in a nonhuman primate with great similarity in gene expression to human.

et al., To provide a temporal framework for the genoarchitecture of brain development, we generated in situ hybridization data for embryonic and postnatal mouse brain at seven developmental stages for ∼2,100 genes, which were processed with an automated informatics pipeline and manually annotated. This resource comprises 434,946 images, seven reference atlases, an ontogenetic ontology, and tools to explore coexpression of genes across neurodevelopment. Gene sets coinciding with developmental phenomena were identified. A temporal shift in the principles governing the molecular organization of the brain was detected, with transient neuromeric, plate-based organization of the brain present at E11.5 and E13.5. Finally, these data provided a transcription factor code that discriminates brain structures and identifies the developmental age of a tissue, providing a foundation for eventual genetic manipulation or tracking of specific brain structures over development. The resource is available as the Allen Developing Mouse Brain Atlas (http://developingmouse.brain-map.org).

SummaryAlthough there have been major advances in elucidating the functional biology of the human brain, relatively little is known of its cellular and molecular organization. Here we report a large-scale characterization of the expression of ∼1,000 genes important for neural functions by in situ hybridization at a cellular resolution in visual and temporal cortices of adult human brains. These data reveal diverse gene expression patterns and remarkable conservation of each individual gene's expression among individuals (95%), cortical areas (84%), and between human and mouse (79%). A small but substantial number of genes (21%) exhibited species-differential expression. Distinct molecular signatures, comprised of genes both common between species and unique to each, were identified for each major cortical cell type. The data suggest that gene expression profile changes may contribute to differential cortical function across species, and in particular, a shift from corticosubcortical to more predominant corticocortical communications in the human brain.

Sequencing of the Leishmania major Friedlin genome is well underway with chromosome 1 (Chr1) and Chr3 having been completely sequenced, and Chr4 virtually complete. Sequencing of several other chromosomes is in progress and the complete genome sequence may be available as soon as 2003. A large proportion (≈ 70%) of the newly identified genes remains unclassified, with many of these being potentially Leishmania (or kinetoplastid-) specific. Most interestingly, the genes are organized into large (> 100–300 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chrl contains two such clusters organized in a ‘divergent’ manner, i.e. the mRNAs for the two sets of genes are both transcribed towards the telomeres. Chr3 contains two ‘convergent’ clusters, with a single ‘divergent’ gene at one telomere, with the two large clusters separated by a tRNA gene. We have characterized several genes from the LD1 (Leishmania DNA 1) region of Chr35. BT1 (formerly ORFG) encodes a biopterin transporter and ORFF encodes a nuclear protein of unknown function. Immunization of mice with recombinant antigens from these genes results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.