High-resolution gene expression atlases for adult and developing mouse brain and spinal cord

Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org).     

If I only had a brain: exploring mouse brain images in the Allen Brain Atlas

The combination of a powerful well-designed user interface with detailed high-quality data sets can create new possibilities for data exploration and analysis. The Allen Brain Atlas (http://www.brain-map.org) provides a collection of tools for examining a set of images that detail gene expression in the mouse brain. Powerful web-based viewers for individual images and parallel examination of related images interact with an external application for three-dimensional views. The underlying dataset, generated via high-throughput analysis of expression patterns of more than 21,000 genes in adult mouse brains, provides three-dimensional views of gene expression patterns displayed in the context of an anatomical ontology. Facilities for filtering views, saving views of interest, annotating images and sharing views via email support the ongoing process of analysis and provide a model for the future of integrated tools for analysing large image data sets.     

Co-expression Profiling of Autism Genes in the Mouse Brain

Autism spectrum disorder (ASD) is one of the most prevalent and highly heritable neurodevelopmental disorders in humans. There is significant evidence that the onset and severity of ASD is governed in part by complex genetic mechanisms affecting the normal development of the brain. To date, a number of genes have been associated with ASD. However, the temporal and spatial co-expression of these genes in the brain remain unclear. To address this issue, we examined the co-expression network of 26 autism genes from AutDB (http://mindspec.org/autdb.html), in the framework of 3,041 genes whose expression energies have the highest correlation between the coronal and sagittal images from the Allen Mouse Brain Atlas database (http://mouse.brain-map.org). These data were derived from in situ hybridization experiments conducted on male, 56-day old C57BL/6J mice co-registered to the Allen Reference Atlas, and were used to generate a normalized co-expression matrix indicating the cosine similarity between expression vectors of genes in this database. The network formed by the autism-associated genes showed a higher degree of co-expression connectivity than seen for the other genes in this dataset (Kolmogorov–Smirnov P = 5×10⁻²⁸). Using Monte Carlo simulations, we identified two cliques of co-expressed genes that were significantly enriched with autism genes (A Bonferroni corrected P<0.05). Genes in both these cliques were significantly over-expressed in the cerebellar cortex (P = 1×10⁻⁵) suggesting possible implication of this brain region in autism. In conclusion, our study provides a detailed profiling of co-expression patterns of autism genes in the mouse brain, and suggests specific brain regions and new candidate genes that could be involved in autism etiology.     

Study of gene function based on spatial co-expression in a high-resolution mouse brain atlas

Background  

The Allen Brain Atlas (ABA) project systematically profiles three-dimensional high-resolution gene expression in postnatal mouse brains for thousands of genes. By unveiling gene behaviors at both the cellular and molecular levels, ABA is becoming a unique and comprehensive neuroscience data source for decoding enigmatic biological processes in the brain. Given the unprecedented volume and complexity of the in situ hybridization image data, data mining in this area is extremely challenging. Currently, the ABA database mainly serves as an online reference for visual inspection of individual genes; the underlying rich information of this large data set is yet to be explored by novel computational tools. In this proof-of-concept study, we studied the hypothesis that genes sharing similar three-dimensional expression profiles in the mouse brain are likely to share similar biological functions.     

Clustering of spatial gene expression patterns in the mouse brain and comparison with classical neuroanatomy

Spatial gene expression profiles provide a novel means of exploring the structural organization of the brain. Computational analysis of these patterns is made possible by genome-scale mapping of the C57BL/6J mouse brain in the Allen Brain Atlas. Here we describe methodology used to explore the spatial structure of gene expression patterns across a set of 3041 genes chosen on the basis of consistency across experimental observations (N = 2). The analysis was performed on smoothed, co-registered 3D expression volumes for each gene obtained by aggregating cellular resolution image data. Following dimensionality and noise reduction, voxels were clustered according to similarity of expression across the gene set. We illustrate the resulting parcellations of the mouse brain for different numbers of clusters (K) and quantitatively compare these parcellations with a classically-defined anatomical reference atlas at different levels of granularity, revealing a high degree of correspondence. These observations suggest that spatial localization of gene expression offers substantial promise in connecting knowledge at the molecular level with higher-level information about brain organization.     

Exogenous ABA induces salt tolerance in indica rice (Oryza sativa L.): The role of OsP5CS1 and OsP5CR gene expression during salt stress

Abscisic acid (ABA) applied exogenously at 100 μM prior to and during the salt-stress period induced salt tolerance in both the salt-susceptible (LPT123) and the genetically related salt-resistant (LPT123-TC171) rice lines, enhanced the survival rate by 20%, and triggered proline (Pro) accumulation earlier than that by salt-stress alone, supporting a role for Pro as an osmoprotectant. In both rice lines, salt-stress induced OsP5CS1 gene expression, suggesting that proline accumulation occurs via OsP5CS1 gene expression during salt stress. An increase in the endogenous ABA level was required for the induction of OsP5CS1 gene expression by salt stress. Under salt stress, topical ABA application-induced OsP5CS1 gene expression only in the salt-resistant line but up-regulated OsP5CR gene expression in both rice lines, suggesting that the increased proline accumulation and salt resistance induced by topical ABA application may result from the up-regulation of OsP5CR and not, directly at least, from OsP5CS1. Moreover, exogenous ABA application up-regulates OsCam1-1 (the salt-stress-responsive calmodulin) gene expression, and calmodulin was shown to play a role in the signal transduction cascade in proline accumulation during salt stress. These data suggest the role of the calmodulin signaling cascade and the induction of OsP5CR gene expression in proline accumulation by exogenous ABA application.     

Comparative gene expression analysis of the engulfment and cell motility (ELMO) protein family in the mouse brain

Engulfment and cell motility (ELMO) proteins bind to Dock180, a guanine nucleotide exchange factor (GEF) of the Rac family, and regulate GEF activity. The resultant ELMO/Dock180/Rac module regulates cytoskeletal reorganization responsible for the engulfment of apoptotic cells, cell migration, and neurite extension. The expression and function of Elmo family proteins in the nervous system, however, are not yet fully understood. Here, we characterize the comparative gene expression profiles of three Elmo family members (Elmo1, Elmo2, and Elmo3) in the brain of C57BL/6J mice, a widely used inbred strain, together with reeler mutant mice to understand gene expression in normal laminated brain areas compared with abnormal areas. Although all three Elmo genes showed widespread mRNA expression over various mouse tissues tested, Elmo1 and Elmo2 were the major types expressed in the brain, and three Elmo genes were up-regulated between the first postnatal week (infant stage) and the third postnatal week (juvenile, weaning stage). In addition, the mRNAs of Elmo genes showed distinct distribution patterns in various brain areas and cell-types; such as neurons including inhibitory interneurons as well as some non-neuronal cells. In the cerebral cortex, the three Elmo genes were widely expressed over many cortical regions, but the predominant areas of Elmo1 and Elmo2 expression tended to be distributed unevenly in the deep (a lower part of the VI) and superficial (II/III) layers, respectively, which also changed depending on the cortical areas and postnatal stages. In the dentate gyrus of the hippocampus, Elmo2 was expressed in dentate granule cells more in the mature stage rather than the immature-differentiating stage. In the thalamus, Elmo1 but not the other members was highly expressed in many nuclei. In the medial habenula, Elmo2 and Elmo3 were expressed at intermediate levels. In the cerebellar cortex, Elmo1 and Elmo2 were expressed in differentiating-mature granule cells and mature granule cells, respectively. In the Purkinje cell layer, Elmo1 and Elmo2 were expressed in Purkinje cells and Bergmann glia, respectively. Disturbed cellular distributions and laminar structures caused by the reeler mutation did not severely change expression in these cell types despite the disturbed cellular distributions and laminar structures, including those of the cerebrum, hippocampus, and cerebellum. Taken together, these results suggested that these three Elmo family members share their functional roles in various brain regions during prenatal-postnatal development.     

Comparative analysis of selenocysteine machinery and selenoproteome gene expression in mouse brain identifies neurons as key functional sites of selenium in mammals

Although dietary selenium (Se) deficiency results in phenotypes associated with selenoprotein depletion in various organs, the brain is protected from Se loss. To address the basis for the critical role of Se in brain function, we carried out comparative gene expression analyses for the complete selenoproteome and associated biosynthetic factors. Using the Allen Brain Atlas, we evaluated 159 regions of adult mouse brain and provided experimental analyses of selected selenoproteins. All 24 selenoprotein mRNAs were expressed in the mouse brain. Most strikingly, neurons in olfactory bulb, hippocampus, cerebral cortex, and cerebellar cortex were exceptionally rich in selenoprotein gene expression, in particular in GPx4, SelK, SelM, SelW, and Sep15. Over half of the selenoprotein genes were also expressed in the choroid plexus. A unique expression pattern was observed for one of the highly expressed selenoprotein genes, SelP, which we suggest to provide neurons with Se. Cluster analysis of the expression data linked certain selenoproteins and selenocysteine machinery genes and suggested functional linkages among selenoproteins, such as that between SelM and Sep15. Overall, this study suggests that the main functions of selenium in mammals are confined to certain neurons in the brain.     

Effects of postharvest storage and dormancy status on ABA content, metabolism, and expression of genes involved in ABA biosynthesis and metabolism in potato tuber tissues

At harvest, and for an indeterminate period thereafter, potato tubers will not sprout and are physiologically dormant. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms controlling ABA content during dormancy as well as the sites of ABA synthesis and catabolism are unknown. As a first step in defining the sites of synthesis and cognate processes regulating ABA turnover during storage and dormancy progression, gene sequences encoding the ABA biosynthetic enzymes zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED) and three catabolism-related genes were used to quantify changes in their relative mRNA abundances in three specific tuber tissues (meristems, their surrounding periderm and underlying cortex) by qRT-PCR. During storage, StZEP expression was relatively constant in meristems, exhibited a biphasic pattern in periderm with transient increases during early and mid-to-late-storage, and peaked during mid-storage in cortex. Expression of two members of the potato NCED gene family was found to correlate with changes in ABA content in meristems (StNCED2) and cortex (StNCED1). Conversely, expression patterns of three putative ABA-8′-hydroxylase (CYP707A) genes during storage varied in a tissue-specific manner with expression of two of these genes rising in meristems and periderm and declining in cortex during storage. These results suggest that ABA synthesis and metabolism occur in all tuber tissues examined and that tuber ABA content during dormancy is the result of a balance of synthesis and metabolism that increasingly favors catabolism as dormancy ends and may be controlled at the level of StNCED and StCYP707A gene activities Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Identification of quantitative trait loci for ABA responsiveness at the seedling stage associated with ABA-regulated gene expression in common wheat

Responsiveness to abscisic acid (ABA) during vegetative growth plays an important role in regulating adaptive responses to various environmental conditions, including activation of a number of ABA-responsive genes. However, the relationship between gene expression and responsiveness to ABA at the seedling stage has not been well studied in wheat. In the present study, quantitative trait locus (QTL) analysis for ABA responsiveness at the seedling stage was performed using recombinant inbred lines derived from a cross between common wheat cultivars showing different ABA responsiveness. Five QTLs were found to be significant, located on chromosomes 1B, 2A, 3A, 6D and 7B. The QTL with the greatest effect was located on chromosome 6D and explained 11.12% of the variance in ABA responsiveness. The other QTLs each accounted for approximately 5–8% of the phenotypic variation. Expression analyses of three ABA-responsive Cor/Lea genes, Wdhn13, Wrab15 and Wrab17, showed that allelic differences in QTLs on chromosomes 2A, 6D and 7B influenced expression of these genes in seedlings treated with ABA. The 3A QTL appeared to be involved in the regulatory system of Wdhn13 and Wrab15, but not Wrab17. The effects of the 2A and 6D QTLs on gene expression were relatively large. The combination of alleles at the QTLs resulted in an additive or synergistic effect on Cor/Lea expression. These results indicate that the QTLs influencing ABA responsiveness are associated with ABA-regulated gene expression and suggest that the QTL on chromosome 6D with the largest effect acts as a key regulator of ABA responses including seedling growth arrest and gene expression during the vegetative stage.     

Laminar and Dorsoventral Molecular Organization of the Medial Entorhinal Cortex Revealed by Large-scale Anatomical Analysis of Gene Expression

Neural circuits in the medial entorhinal cortex (MEC) encode an animal’s position and orientation in space. Within the MEC spatial representations, including grid and directional firing fields, have a laminar and dorsoventral organization that corresponds to a similar topography of neuronal connectivity and cellular properties. Yet, in part due to the challenges of integrating anatomical data at the resolution of cortical layers and borders, we know little about the molecular components underlying this organization. To address this we develop a new computational pipeline for high-throughput analysis and comparison of in situ hybridization (ISH) images at laminar resolution. We apply this pipeline to ISH data for over 16,000 genes in the Allen Brain Atlas and validate our analysis with RNA sequencing of MEC tissue from adult mice. We find that differential gene expression delineates the borders of the MEC with neighboring brain structures and reveals its laminar and dorsoventral organization. We propose a new molecular basis for distinguishing the deep layers of the MEC and show that their similarity to corresponding layers of neocortex is greater than that of superficial layers. Our analysis identifies ion channel-, cell adhesion- and synapse-related genes as candidates for functional differentiation of MEC layers and for encoding of spatial information at different scales along the dorsoventral axis of the MEC. We also reveal laminar organization of genes related to disease pathology and suggest that a high metabolic demand predisposes layer II to neurodegenerative pathology. In principle, our computational pipeline can be applied to high-throughput analysis of many forms of neuroanatomical data. Our results support the hypothesis that differences in gene expression contribute to functional specialization of superficial layers of the MEC and dorsoventral organization of the scale of spatial representations.     

Developmental expression of sorting nexin 3 in the mouse central nervous system

We previously reported that sorting nexin 3 (SNX3), a protein belonging to the sorting nexin family, regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The snx3 gene is disrupted in patients with microcephaly, microphthalmia, ectrodactyly, and prognathism (MMEP) and mental retardation, demonstrating that SNX3 plays an important role in the genesis of these organs during development. The present study was designed to determine the expression pattern of snx3 mRNA, particularly in the mouse central nervous system (CNS), from the embryonic stage to adulthood. Whole mount in situ hybridization of embryonic day (E) 9.5 and 10.5 mouse embryos revealed strong positive signals for snx3 mRNA in the forebrain, pharyngeal arches, eyes, and limb buds. In situ hybridization analyses of embryonic and neonatal brain sections revealed that snx3 mRNA is mainly expressed in the cerebral cortex, hippocampus, piriform cortex, cerebellum, and spinal cord. In adulthood, the expression of snx3 mRNA is observed in the cerebral cortex, hippocampus, piriform cortex, and cerebellar neurons. Thus, snx3 mRNA is expressed during neural development and in adult neural tissues, suggesting that SNX3 may play an important role in the development and function of the CNS.