Scientific Understanding of Consciousness
Brain Transcriptome Atlas
Nature 489, 391–399 (20 September 2012)
An anatomically comprehensive atlas of the adult human brain transcriptome
Michael J. Hawrylycz, et al.
Allen Institute for Brain Science, Seattle, Washington 98103, USA; et al.
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.
The enormous complexity of the human brain is a function of its precise circuitry, its structural and cellular diversity, and, ultimately, the regulation of its underlying transcriptome. In rodents, brain- and transcriptome-wide, cellular-resolution maps of transcript distributions are widely useful resources to complement genomic sequence data. However, owing to the challenges of a 1,000-fold increase in size from mouse to human, limitations in post-mortem tissue availability and quality, and the destructive nature of molecular assays, there has been no human counterpart so far. Several important recent studies have begun to analyse transcriptional dynamics during human brain development, although only in a small number of relatively coarse brain regions. Characterizing the complete transcriptional architecture of the human brain will provide important information for understanding the impact of genetic disorders on different brain regions and functional circuits. Furthermore, conservation and divergence in brain function between humans and other species provide essential information for the understanding of drug action, which is often poorly conserved across species.
The goal of the Allen Human Brain Atlas is to create a comprehensive map of transcript usage across the entire adult brain, with the emphasis on anatomically complete coverage at a fine nuclear resolution in a small number of high-quality, clinically unremarkable brains profiled with DNA microarrays for quantitative gene-level transcriptome coverage. Furthermore, structural brain imaging data were obtained from each individual to visualize gene expression data in its native three-dimensional anatomical coordinate space, and to allow correlations between imaging and transcriptome modalities. These data are freely accessible via the Allen Brain Atlas data portal (http://www.brain-map.org).
Global mapping of transcript distributions
A tissue processing and data collection pipeline was established to image the brain and subsequently dissect tissue samples from approximately 900 anatomically defined sites for RNA isolation and microarray analysis. Two complete normal male brains were analysed from donors aged 24 and 39 years.
Interestingly, no statistically significant hemispheric differences could be identified at this fine structural level that were corroborated in both brains. Although surprising given well described lateralization of function, this finding is consistent with a recent study of developing human neocortex that failed to identify hemispheric differences despite extensive efforts using microarrays and quantitative PCR. It may be that the basis for lateralization of function involves more subtle changes in specific cellular components, differences in relative area rather than type of functional domains between hemispheres, or is more related to functional connectivity patterns than molecular differentiation.
Global transcriptional architecture of the adult brain
Most genes with high variation across brain regions are not selective for a single major brain region; rather, they are expressed in multiple regions and non-uniformly within these regions. This suggests that many genes may be quite pleiotropic with respect to brain function, and that local gene regulation in specific cytoarchitectural nuclei is the most important level of resolution. To summarize the complexity of structural variation and examine the extent to which major brain regions display local enrichment in specific fine cytoarchitectural divisions, we created a specificity index for each major region that measures enrichment in subdivisions of that region. The index measures transcriptional diversity within regions. The neocortex and cerebellum display the least internal heterogeneity. In contrast, subcortical regions with many well-defined nuclei show the greatest local heterogeneity, including the myelencephalon, mesencephalon, pons, hippocampus and hypothalamus. It is also possible to identify genes with either brain-wide (global) or within structure (local) ubiquity. Not surprisingly, these gene sets are enriched for cellular organelles and ‘housekeeping’ functions (for example, ribosome, mitochondrion, metabolism).
Local patterning reflects hippocampal cytoarchitecture
To explore local variation, we identified unique transcriptional signatures by analysis of variance (ANOVA) for the hippocampus. Following unsupervised hierarchical 2D clustering, cytoarchitecturally discrete subdivisions of the hippocampus (dentate gyrus, CA fields and subiculum) showed distinctive expression patterns sufficiently robust to cluster together like-samples while distinguishing subdivisions from one another. Interestingly, samples from the CA3 and CA4 subfields were not discriminable consistent with the view that CA4 is not a functionally distinct subfield from CA3. Similarly robust regional clustering was observed in the mesencephalon, pons and myelencephalon.
Our extensive neocortical sampling allowed us to investigate transcriptional variation across the neocortex in relation to spatial position and functional parcellation. Although highly differential expression between cortical regions is much less pronounced than between other brain regions, many genes show statistically significant variation between lobes or gyri at a lower threshold. We first identified the 1,000 genes displaying the most significant variation in expression between 56 gyri in both brains. We then performed principal component analysis (PCA) on the 1,000 (genes) by 56 (sampled gyri) matrices for both brains. The first three principal components had striking selectivity for specific cortical regions (samples ordered by lobe and roughly rostral to caudal within each lobe) and were generally reproducible across both brains. PC1 is associated with primary sensorimotor cortices, with relative differential expression in precentral (motor) and postcentral (somatosensory) cortex, Heschl’s gyrus (primary auditory) and primary and secondary visual areas. Confirmation of the visual cortex enrichment by ISH for several synaptic transmission-associated genes highly correlated to PC1. PC2 has areal selectivity for posterior orbital, paraolfactory and subcallosal gyri in the frontal lobe, the temporal pole, and the primary visual cortex. PC3 is primarily differential in frontal cortex compared to temporal and occipital cortex. These first three components accounted for a large amount of the variance. The spatial organization of the first three principal components was highly correlated between brains.
Regional transcriptional signatures highly conserved
Molecular studies of human tissues are necessary for understanding the details of human brain function in the context of specific pathways and cell types and how they are affected in disease conditions.
Regional transcriptional signatures are highly conserved between the two brains assayed. These two individuals were males of similar age and ethnicity and therefore do not capture population or sex diversity; nevertheless, this high degree of similarity is suggestive of a strong underlying common blueprint for the human brain transcriptome and is consistent with other recent studies of human neocortical gene expression. The availability of an entire hemisphere of a third brain specimen enabled several confirmatory analyses to be performed. In particular, current results report positively on the network analyses, structural variation of gene expression, and genetic topography of the neocortex. In summary, the high recapitulation of gene expression patterns across all three brains indicates that the basic transcriptional blueprint is robust across individuals. Ongoing work is focused on processing additional brains of both sexes to estimate the consistency of this blueprint.
The primary feature that distinguishes the human brain from that of other species is the enormous expansion of the neocortex relative to total brain volume. Our extensive profiling allowed us to ask directly how transcription varies across the neocortex. Surprisingly, we find a remarkable degree of transcriptional uniformity compared to other brain regions, apparently reflecting the similarity in laminar architecture across the entire neocortex. However, there is significant, albeit less robust, variation in gene expression across cortical areas with two hallmark features. First, individual cortical samples showed such strong transcriptional similarities to neighbouring samples that the topography of the neocortex as a whole can, in part, be reconstructed based on their molecular profiles. One possible explanation is that these proximity relationships mirror lineage relationships of neocortical neurons generated from proximal parts of the developing neuroepithelium. Second, some primary sensory and motor regions do have distinct whole-transcriptome signatures, probably related to their specialized cellular and functional architecture. It is also likely that other more subtle features of cortical parcellation may not have been detected in the current analysis, including those identified using neurotransmitter receptor distributions and functional connectivity. One issue is that gyral patterns do not correlate perfectly with either cytoarchitectural or functional cortical parcellation. Greater regional differences may emerge if the samples can be grouped either by Brodmann area or on the basis of correlation to functional parcellations derived from functional imaging studies, now possible given the mapping of these data to MRI coordinates. Furthermore, it is likely that greater variation across areas will be found when assayed at the level of specific cortical cell types, as the excitatory neuron types in different layers display highly distinct molecular profiles that have been shown to vary significantly across areas in primate neocortex. Finally, higher confidence in consistent regional differences should emerge as more samples are investigated. Nevertheless, the relative homogeneity of the two largest neuronal structures, with ~69 billion (cerebellar cortex) and ~16 billion (cortex) neurons out of the 86 billion neurons in the human brain, is striking and suggests an evolutionary expansion of a canonical cortical blueprint.
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