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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.

Schizophrenia is highly heritable, and genetic risk for vulnerability is conferred by a large number of alleles, including common alleles of small effect that have been detected through genome-wide association. Because the public health impact of schizophrenia is so substantial, data and tools to further our study of this complex disease are needed. The Allen Brain Atlas ( www.brain-map.org ) is an online, publicly available resource that integrates gene expression and connectivity data with neuroanatomical information for mouse, human, and nonhuman primate, and it may be of interest to researchers working in these areas. As one of the most comprehensive gene expression resources for the nervous system, scientists regularly use these data to learn about the expression profile of genes in various neuroanatomical regions. Additional uses include searching for biomarkers, correlating gene expression to anatomy, and other large-scale correlative data analysis. This chapter reviews the resources available and describes how certain ones may be of use to those studying schizophrenia.

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.

available in PMC 2016 March 04, An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity., National Institutes of Health (U.S.) (NIH grant DA028298), Wellcome Trust (London, England) (Grant), National Institutes of Health (U.S.) (NIH grant MH085500)

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.

Background Autism involves early brain overgrowth and dysfunction, which is most strongly evident in the prefrontal cortex. As assessed on pathological analysis, an excess of neurons in the prefrontal cortex among children with autism signals a disturbance in prenatal development and may be concomitant with abnormal cell type and laminar development. Methods To systematically examine neocortical architecture during the early years after the onset of autism, we used RNA in situ hybridization with a panel of layer- and cell-type– specific molecular markers to phenotype cortical microstructure. We assayed markers for neurons and glia, along with genes that have been implicated in the risk of autism, in prefrontal, temporal, and occipital neocortical tissue from postmortem samples obtained from children with autism and unaffected children between the ages of 2 and 15 years. Results We observed focal patches of abnormal laminar cytoarchitecture and cortical disorganization of neurons, but not glia, in prefrontal and temporal cortical tissue from 10 of 11 children with autism and from 1 of 11 unaffected children. We observed heterogeneity between cases with respect to cell types that were most abnormal in the patches and the layers that were most affected by the pathological features. No cortical layer was uniformly spared, with the clearest signs of abnormal expression in layers 4 and 5. Three-dimensional reconstruction of layer markers confirmed the focal geometry and size of patches. Conclusions In this small, explorative study, we found focal disruption of cortical laminar architecture in the cortexes of a majority of young children with autism. Our data support a probable dysregulation of layer formation and layer-specific neuronal differentiation at prenatal developmental stages. (Funded by the Simons Foundation and others.)

During human brain development from fetal stage to adulthood, the white matter (WM) tracts undergo dramatic changes. Diffusion tensor imaging (DTI), a widely used magnetic resonance imaging (MRI) modality, offers insight into the dynamic changes of WM fibers as these fibers can be noninvasively traced and three-dimensionally (3D) reconstructed with DTI tractography. The DTI and conventional T1 weighted MRI images also provide sufficient cortical anatomical details for mapping the cortical regions of interests (ROIs). In this paper, we described basic concepts and methods of DTI techniques that can be used to trace major WM tracts noninvasively from fetal brain of 14 postconceptional weeks (pcw) to adult brain. We applied these techniques to acquire DTI data and trace, reconstruct and visualize major WM tracts during development. After categorizing major WM fiber bundles into five unique functional tract groups, namely limbic, brain stem, projection, commissural and association tracts, we revealed formation and maturation of these 3D reconstructed WM tracts of the developing human brain. The structural and connectional imaging data offered by DTI provides the anatomical backbone of transcriptional atlas of the developing human brain.

The Allen Brain Atlas is an online publicly available resource that integrates gene expression and connectivity data with neuroanatomical information for the mouse, human, and non-human primate. Launched in 2004 by the Allen Institute for Brain Science, the portal currently receives about 45000 unique users each month. More than one petabyte of in situ hybridization imagery and over 240 million microarray data points from six adult human brains representing 3700 tissue samples have been generated to date. As one of the most comprehensive gene expression resources for the nervous system, scientists regularly use these resources to study the expression profile of genes in the various regions of the brain. Additional usage includes searching for biomarkers, correlating gene expression to neuroanatomy, and other large-scale correlative data analysis. This chapter reviews the resources available and describes how they were constructed to enable development of visualization and search tools to analyze the massive amount of data generated. Finally, examples are provided on how these tools can be leveraged for scientific discovery.

Detailed anatomical understanding of the human brain is essential for unraveling its functional architecture, yet current reference atlases have major limitations such as lack of whole‐brain coverage, relatively low image resolution, and sparse structural annotation. We present the first digital human brain atlas to incorporate neuroimaging, high‐resolution histology, and chemoarchitecture across a complete adult female brain, consisting of magnetic resonance imaging (MRI), diffusion‐weighted imaging (DWI), and 1,356 large‐format cellular resolution (1 µm/pixel) Nissl and immunohistochemistry anatomical plates. The atlas is comprehensively annotated for 862 structures, including 117 white matter tracts and several novel cyto‐ and chemoarchitecturally defined structures, and these annotations were transferred onto the matching MRI dataset. Neocortical delineations were done for sulci, gyri, and modified Brodmann areas to link macroscopic anatomical and microscopic cytoarchitectural parcellations. Correlated neuroimaging and histological structural delineation allowed fine feature identification in MRI data and subsequent structural identification in MRI data from other brains. This interactive online digital atlas is integrated with existing Allen Institute for Brain Science gene expression atlases and is publicly accessible as a resource for the neuroscience community. J. Comp. Neurol. 524:3127–3481, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

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.

A new family of Pneumocystis carinii genes (called MSR for MSG-related) that encodes peptides related to the major surface glycoprotein (MSG) is described. Members of the MSR sequence family are linked to MSG genes and are located near the ends of at least 13 chromosomes. Transcripts encoding different MSR isoforms were present in a single population of P. carinii f. sp. carinii, showing that multiple MSR genes were expressed. Two size classes of MSR mRNA, 2.4 and 3.5 kb, were detected. Both sizes of MSR mRNA lacked the upstream conserved sequence (UCS), which is found on the 5' end of MSG mRNAs because MSG genes must be linked to the UCS to be transcribed. The absence of the UCS from MSR mRNAs suggests that expression of MSR genes does not require linkage to the UCS locus.

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.

The surface of Pneumocystis carinii sp. f. carinii contains an antigen known as major surface glycoprotein (MSG), which is encoded by about 100 heterogeneous genes. Expression of MSG genes is not well understood. Previous work identified a sequence termed UCS, which is present at the beginning of nearly all MSG mRNAs, and which is likely to be involved in regulation of MSG gene transcription. Here we show that the UCS was present in one copy per haploid genome, but that different MSG genes were linked to the unique UCS locus in different members of a P. c. carinii population, predicting that individual organisms transcribe a limited number of MSG genes. This prediction was supported by indirect immunofluorescence observations. Comparison of three different populations of P. c. carinii showed that each contained a different set of MSG genes linked to the UCS, suggesting that UCS-MSG junctions are formed by recombination during population growth. Both the UCS and MSG genes were shown to be located at the ends of chromosomes, suggesting that the mechanism for UCS-MSG recombination is reciprocal exchange.

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.

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.

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

Sleep deprivation (SD) leads to a suite of cognitive and behavioral impairments, and yet the molecular consequences of SD in the brain are poorly understood. Using a systematic immediate-early gene (IEG) mapping to detect neuronal activation, the consequences of SD were mapped primarily to forebrain regions. SD was found to both induce and suppress IEG expression (and thus neuronal activity) in subregions of neocortex, striatum, and other brain regions. Laser microdissection and cDNA microarrays were used to identify the molecular consequences of SD in seven brain regions. In situ hybridization (ISH) for 222 genes selected from the microarray data and other sources confirmed that robust molecular changes were largely restricted to the forebrain. Analysis of the ISH data for 222 genes (publicly accessible at http://sleep.alleninstitute.org) provided a molecular and anatomic signature of the effects of SD on the brain. The suprachiasmatic nucleus (SCN) and the neocortex exhibited differential regulation of the same genes, such that in the SCN genes exhibited time-of-day effects while in the neocortex, genes exhibited only SD and waking (W) effects. In the neocortex, SD activated gene expression in areal-, layer-, and cell type-specific manner. In the forebrain, SD preferentially activated excitatory neurons, as demonstrated by double-labeling, except for striatum which consists primarily of inhibitory neurons. These data provide a characterization of the anatomical and cell type-specific signatures of SD on neuronal activity and gene expression that may account for the associated cognitive and behavioral effects.

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.

Susan M. Sunkin and James R. Stringer*Department of Molecular Genetics, Biochemistry andMicrobiology, College of Medicine, University ofCincinnati, Cincinnati, OH 45267-0524, USA.SummaryThe major surface glycoprotein (MSG) of P. carinii f.sp. cariniiis a family of proteins encoded by a familyof heterogeneous genes. Messenger RNAs encodingdifferent MSG isoforms start with the same sequence,called the upstream conserved sequence (UCS), whichis encoded by a single locus. The mechanism by whichthe UCS becomes part of different MSG mRNAs is notobvious becauseatleast15loci, which aredistributedthroughout the genome, encode MSGs. One possibi-lity is that attachment to the UCS locus is requiredfor the transcription of an MSG gene. The alternativeto this expression site model is that mRNAs acquirethe UCS by RNA splicing. To distinguish betweenthese two models, UCS/MSG junctions in the genomewere compared with UCS/MSG junctions in mRNA.The UCS/MSG junctions in the mRNA matched thosein the genome, as would be expected if splicing didnot contribute to the attachment of the UCS to the 58ends of MSG mRNAs. Given that few if any MSGmRNAs lack the UCS, the correspondence betweenthe UCS/MSG junctions in transcripts and those inthe genome indicates that attachment to the UCS isboth necessary and sufficient for transcription of anMSG gene.IntroductionPneumocystis carinii is a fungus that can cause pneu-monia in immunocompromised humans (Walzer et al.,1974; Mills, 1986). Other mammals can also be infectedby P. carinii, but each host species harbours a differentgenetic variety of the microbe (Sinclair et al., 1991; Edlindet al., 1992; Stringer, 1993; J.R. Stringer et al., 1993; Dei-Cas et al., 1994; De Luca et al., 1994; Keely et al., 1994;Peters et al., 1994a,b; Weinberg et al., 1994). The geneticdivergence among these varieties is substantial, but defi-nitive evidence of multiple species of Pneumocystis islacking. Accordingly, the term formae specialis(meaningspecial form) is used to designate the varieties of P.carinii(Pneumocystis Workshop, 1994). None of the spe-cialformsofP.cariniigrowswellenoughinculturetoallowpropagation of clonally derived stocks, but large numbersof organisms can be obtained routinely from laboratoryanimals. The organisms used in this study were obtainedfrom immunosuppressed rats that were infected with P.carinii formae specialis carinii(P. carinii f. sp. carinii)byexposure to airborne organisms produced by other immu-nosuppressed infected rats (Frenkel et al., 1966; Arm-strong and Cushion, 1993; Cushion et al., 1993).All studied special forms of P. cariniiare coated with amajor surface glycoprotein (MSG) (Walzer and Linke,1987; Kovacs et al., 1988; Pesanti and Shanley, 1988;Linke and Walzer, 1989; Radding et al., 1989; Tanabe etal., 1989; Lundgren et al., 1991), also known as gpA(Gigliotti et al., 1988; Gigliotti, 1992). In P. cariniispecialforms recovered from rats, ferrets, mice and humans, ithas been shown that MSG is actually a family of proteinsencoded by a family of heterogeneous genes (S.L. Strin-ger et al., 1991; 1993; Haidaris et al., 1992; Kovacs etal., 1993; Garbe and Stringer, 1994; Wright et al., 1994).The genome of P. carinii f. sp. carinii contains approxi-mately 100 different MSG genes (Stringer et al., 1991),which are organized in clusters that are located at theends of each of the 15 chromosomes (Wada et al., 1993;Sunkin et al., 1994; Sunkin and Stringer, 1996; Underwoodet al., 1996; Wada and Nakamura, 1996). Populations ofP. carinii f. sp. carinii contain mRNAs encoding multipleMSG isoforms (Linke et al., 1994), but expression of thisgene family within an individual organism is thought to belimited, based on both phenotypic and molecular geneticdata. Phenotypic data indicating limited expression includeindirect immunofluorescence studies that showed thatnot all organisms within a population could be labelledwith an antibody directed against a subset of MSG iso-forms (Angus et al., 1996; Sunkin and Stringer, 1996). Inaddition,thefractionof organismslabelledbysuchananti-body varied among populations(Angus et al., 1996). Addi-tional phenotypic evidence of limited MSG expression hasbeenprovidedbyWesternblotting, whichshowedthat someP. carinii f. sp. carinii populations contained a particular

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.

This study introduces a novel method for standardized relative quantification of colorimetric in situ hybridization signal that enables a large-scale cross-platform expression level comparison of in situ hybridization with two publicly available microarray brain data sources., With the emergence of genome-wide colorimetric in situ hybridization (ISH) data sets such as the Allen Brain Atlas, it is important to understand the relationship between this gene expression modality and those derived from more quantitative based technologies. This study introduces a novel method for standardized relative quantification of colorimetric ISH signal that enables a large-scale cross-platform expression level comparison of ISH with two publicly available microarray brain data sources.