Scientific Understanding of Consciousness
Embryonic Brain Development Studies via Prenatal Transcription Processes
Nature 508, 199–206 (10 April 2014)
Transcriptional landscape of the prenatal human brain
Jeremy A. Miller, et.al.
Allen Institute for Brain Science, Seattle, Washington 98103, USA
Division of Genetic Medicine, Department of Pediatrics, University of Washington, 1959 North East Pacific Street, Box 356320, Seattle, Washington 98195, USA
Department of Radiology, Harvard Medical School, Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
Computer Science and AI Lab, MIT, Cambridge, Massachusetts 02139, USA
Department of Neurobiology and Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, Connecticut 06510, USA
Program in Computational Biology and Bioinformatics, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA
Department of Computer Science, Yale University, New Haven, Connecticut 06520, USA
Program in Neurogenetics, Department of Neurology and Semel Institute David Geffen School of Medicine, UCLA, Los Angeles, California 90095, USA
Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, Washington 98101, USA
Department of Neurological Surgery, University of Washington School of Medicine, Seattle, Washington 98105, USA
Advanced Imaging Research Center, UT Southwestern Medical Center, Dallas, Texas 75390, USA
Zilkha Neurogenetic Institute, and Department of Psychiatry, University of Southern California, Los Angeles, California 90033, USA
Department of Pediatrics, Children’s Hospital, Los Angeles, California 90027, USA
Keck School of Medicine, University of Southern California, Los Angeles, California 90089, USA
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.
The human brain develops following a complex, highly stereotyped series of histogenic events that depend on regulated differential gene expression, and acquired or inherited disruption can lead to devastating consequences. Mainly due to limitations in access to human prenatal tissue, most developmental studies are performed in mouse or non-human primates. Yet significant species differences exist, necessitating the study of human brain. For example, the human neocortex has undergone massive evolutionary expansion, particularly in superficial layers, probably due to differences in rates of progenitor pool expansion during neurogenesis compared to other species. A secondary progenitor zone, the subventricular zone (SZ) is present in all mammals, but is split into an outer and inner region in primates. The transient subplate zone (SP) is greatly expanded in human as is the subpial granular zone (SG), a transient compartment at the pial surface composed primarily of tangentially migrating neurons. Furthermore, there is evidence for species differences in the developmental origin of cortical GABAergic interneurons. In mouse, nearly all originate from the striatal ganglionic eminences (GEs) of the ventral telencephalon; however, the origin of human cortical interneurons remains controversial. Finally, understanding the emergence of cortical specialization for language can only be studied in humans.
Recent studies have begun to analyse the developing brain and neocortical transcriptome. Profiling of mid-gestational human brain identified many genes differentially expressed between major regions, including genes associated with human-accelerated conserved noncoding sequences (haCNSs). Gene expression also varies between cell populations, and more detailed analysis of layers of fetal mouse neocortex found >2,500 genes differentially expressed between ventricular zone (VZ), subventricular zone, intermediate zone (IZ), and cortical plate (CP). Species differences in distinct fetal transient zones, including the subventricular zone, subplate zone, cortical plate, and subpial granular zone, have also been described.
The goal of the current project was to create resources for studying prenatal human brain development and the early roots of neurodevelopmental and psychiatric disorders. These include anatomical reference atlases similar to those for model organisms, and an anatomically comprehensive, detailed transcriptional profiling of normal mid-gestational brain modelled on atlases of adult mouse and human brain and using methods for selective analysis of discrete structural nuclei and layers. These data are freely accessible as part of the BrainSpan Atlas of the Developing Human Brain via the Allen Brain Atlas data portal.
Four intact high-quality mid-gestational brains, two from 15 and 16 post-conceptual weeks (pcw) and two from 21 pcw were used to create detailed de novo reference atlases and transcriptome data sets. The entire left hemisphere of each specimen was coronally, serially cryosectioned onto polyethylene naphthalate (PEN) membrane slides for laser microdissection (LMD), with interleaved slides for histological staining (Nissl, acetylcholinesterase (AChE), and in situ hybridization for GAP43) for detailed structure identification. Approximately 300 anatomical regions per specimen were isolated for RNA isolation, amplification and microarray analysis on custom 64K Agilent microarrays. For one 15 pcw and one 21 pcw specimen, the right hemisphere was processed similarly but used for in situ hybridization and Nissl staining. These data were anatomically delineated to make digital reference atlases.
We assayed ~25 areas of the developing neocortex, delineating nine layers per area (here referring to fetal mitotic and post-mitotic zones and not layers 1–6 of mature neocortex):
Different layers show robust and unique molecular signatures, and samples group by layer using hierarchical clustering.
To identify principal features of the developing cortical transcriptome, we performed weighted gene co-expression network analysis (WGCNA) on all 526 neocortical samples, and identified 42 modules of co-expressed genes. WGCNA clusters genes with similar expression patterns in an unbiased manner, allowing a biological interpretation of transcriptional patterns.
Germinal layers contain various cortical progenitors including radial glia in the ventricular zone, intermediate progenitors (IP) in the SZi, and ORG in the SZo and these radial glia may be quite diverse.
The subplate is a largely transient zone beneath the cortical plate that plays an important role in establishment of thalamocortical connectivity. Subplate generation is protracted in primates, and its thickness particularly expanded in human. In mouse and other species this layer is molecularly distinct, and our laminar profiling also identified many subplate-enriched genes in human.
Developmental gradients in neocortex
Cortical patterning is likely a result of intrinsic signalling, controlled in part by graded expression of transcription factors during early cortical development, followed by extrinsic signalling from thalamic afferents after the start of corticogenesis.
Conserved non-coding sequences (CNSs) are genomic regions with exceptionally high similarity across divergent organisms, and therefore thought to be important for organism viability. CNSs are typically located by genes important for developmental regulation, and many show significant enhancer activity in brain. Genes near CNSs with significantly accelerated rates of substitution in the human lineage (haCNSs) are particularly likely to show differential expression between regions of developing human neocortex, indicating transcriptional regulation by haCNSs may be important in human-specific neurodevelopment.
Studies of the developing human brain are essential for elucidating the details of human brain formation, function and evolutionary differences, and for understanding developmental mechanisms underlying neurodevelopmental disorders such as autism and schizophrenia. The atlas of the mid-gestational human brain described here, part of the BrainSpan Atlas of the Developing Human Brain, builds on digital molecular brain atlasing efforts in mouse and adult human by providing transcriptome resources on prenatal specimens typically inaccessible for research. Several recent studies have assayed a limited set of brain structures and layers from prenatal human brain. In contrast, the current project aimed for anatomical comprehensiveness at a fine nuclear/laminar level, albeit with a small number of specimens. This degree of specificity necessitated using available methods for small sample amplification and DNA microarrays (the same platform recently used for adult human), but newer techniques may soon allow moving to the resolution of single cells using RNA sequencing for complete transcriptome coverage.
Many differences in cortical development between human, non-human primate and rodent have been documented, including an expanded subplate zone and subpial granular zone, expansion of association areas particularly in frontal lobe, expansion of superficial layers that greatly increase the extent of cortico-cortical connections, and the appearance of a secondary proliferative zone, the SZo, that probably allow the massive expansion of human cortex. We find transcriptional features related to each of these anatomical features, although we were able to identify only minimal molecular differences between the inner and outer subventricular zones, leaving open the question of what distinguishes this primate-specific zone of cortical precursors. These data also provide a powerful map to pin an anatomical and developmental locus on genes related to neurodevelopmental disease origins and human-specific brain function and evolution. Although the current analyses only scratch the surface, these data will be extremely useful for generating and testing new hypotheses about molecular substrates for specific features of human brain development and function.
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