Molecular features driving cellular complexity of human brain evolution.

Human-specific genomic changes contribute to the unique functionalities of the human brain1-5. The cellular heterogeneity of the human brain6,7 and the complex regulation of gene expression highlight the need to characterize human-specific molecular features at cellular resolution. Here we analysed single-nucleus RNA-sequencing and single-nucleus assay for transposase-accessible chromatin with sequencing datasets for human, chimpanzee and rhesus macaque brain tissue from posterior cingulate cortex. We show a human-specific increase of oligodendrocyte progenitor cells and a decrease of mature oligodendrocytes across cortical tissues. Human-specific regulatory changes were accelerated in oligodendrocyte progenitor cells, and we highlight key biological pathways that may be associated with the proportional changes. We also identify human-specific regulatory changes in neuronal subtypes, which reveal human-specific upregulation of FOXP2 in only two of the neuronal subtypes. We additionally identify hundreds of new human accelerated genomic regions associated with human-specific chromatin accessibility changes. Our data also reveal that FOS::JUN and FOX motifs are enriched in the human-specifically accessible chromatin regions of excitatory neuronal subtypes. Together, our results reveal several new mechanisms underlying the evolutionary innovation of human brain at cell-type resolution.

Evolutionary neurogenomics at single-cell resolution.

The human brain is composed of increasingly recognized heterogeneous cell types. Applying single-cell genomics to brain tissue can elucidate relative cell type proportions as well as differential gene expression and regulation among humans and other species. Here, we review recent studies that utilized high-throughput genomics approaches to compare brains among species at single-cell resolution. These studies identified genomic elements that are similar among species as well as evolutionary novelties on the human lineage. We focus on those human-relevant innovations and discuss the biological implications of these modifications. Finally, we discuss areas of comparative single-cell genomics that remain unexplored either due to needed technological advances or due to biological availability at the brain region or species level.

Spatial Single-cell Analysis Decodes Cortical Layer and Area Specification.

The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas1,2. The layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization3,4. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation5,6,7,8. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based cell segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing 16 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable in the laminar distribution of excitatory neuronal subtypes by mid-gestation, long before the emergence of cytoarchitectural layers. Notably, while anterior-posterior gradients of neuronal subtypes were generally observed in most cortical areas, a striking exception was the sharp molecular border between primary (V1) and secondary visual cortices (V2) at gestational week 20. Here we discovered an abrupt binary shift in neuronal subtype specification at the earliest stages, challenging the notion that continuous morphogen gradients dictate mid-gestation cortical arealization6,10. Moreover, integrating single-nuclei RNA-sequencing and in situ whole transcriptomics revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, suggesting a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This work not only provides a valuable resource for the field, but also establishes a spatially resolved single-cell analysis paradigm that paves the way for a comprehensive developmental atlas of the human brain.

Decoding DNA sequence-driven evolution of the human brain epigenome at cellular resolution.

DNA-based evolutionary comparisons of regulatory genomic elements enable insight into functional changes, overcoming tissue inaccessibility. Here, we harnessed adult and fetal cortex single-cell ATAC-seq datasets to uncover DNA substitutions specific to the human and human-ancestral lineages within apes. We found that fetal microglia identity is evolutionarily divergent in all lineages, whereas other cell types are conserved. Using multiomic datasets, we further identified genes linked to multiple lineage-divergent gene regulatory elements and implicated biological pathways associated with these divergent features. We also uncovered patterns of transcription factor binding site evolution across lineages and identified expansion of bHLH-PAS factor targets in human-hominin lineages, and MEF2 factor targets in the ape lineage. Finally, conserved features were more enriched in brain disease variants, whereas there was no distinct enrichment on the human lineage compared to its ancestral lineages. Our study identifies major evolutionary patterns in the human brain epigenome at cellular resolution.

Neuronal ambient RNA contamination causes misinterpreted and masked cell types in brain single-nuclei datasets.

Ambient RNA contamination in single-cell and single-nuclei RNA sequencing (snRNA-seq) is a significant problem, but its consequences are poorly understood. Here, we show that ambient RNAs in brain snRNA-seq datasets have a nuclear or non-nuclear origin with distinct gene set signatures. Both ambient RNA signatures are predominantly neuronal, and we find that some previously annotated neuronal cell types are distinguished by ambient RNA contamination. We detect pervasive neuronal ambient RNA contamination in all glial cell types unless glia and neurons are physically separated prior to sequencing. We demonstrate that this contamination can be removed in silico and show that previous single-nuclei RNA-seq-based annotations of immature oligodendrocytes are glial nuclei contaminated with ambient RNAs. After ambient RNA removal, we detect rare, committed oligodendrocyte progenitor cells not annotated in most previous adult human brain datasets. Together, these results provide an in-depth analysis of ambient RNA contamination in brain single-nuclei datasets.

Association between resting-state functional brain connectivity and gene expression is altered in autism spectrum disorder.

Gene expression covaries with brain activity as measured by resting state functional magnetic resonance imaging (MRI). However, it is unclear how genomic differences driven by disease state can affect this relationship. Here, we integrate from the ABIDE I and II imaging cohorts with datasets of gene expression in brains of neurotypical individuals and individuals with autism spectrum disorder (ASD) with regionally matched brain activity measurements from fMRI datasets. We identify genes linked with brain activity whose association is disrupted in ASD. We identified a subset of genes that showed a differential developmental trajectory in individuals with ASD compared with controls. These genes are enriched in voltage-gated ion channels and inhibitory neurons, pointing to excitation-inhibition imbalance in ASD. We further assessed differences at the regional level showing that the primary visual cortex is the most affected region in ASD. Our results link disrupted brain expression patterns of individuals with ASD to brain activity and show developmental, cell type, and regional enrichment of activity linked genes.

Gene-expression correlates of the oscillatory signatures supporting human episodic memory encoding.

In humans, brain oscillations support critical features of memory formation. However, understanding the molecular mechanisms underlying this activity remains a major challenge. Here, we measured memory-sensitive oscillations using intracranial electroencephalography recordings from the temporal cortex of patients performing an episodic memory task. When these patients subsequently underwent resection, we employed transcriptomics on the temporal cortex to link gene expression with brain oscillations and identified genes correlated with oscillatory signatures of memory formation across six frequency bands. A co-expression analysis isolated oscillatory signature-specific modules associated with neuropsychiatric disorders and ion channel activity, with highly correlated genes exhibiting strong connectivity within these modules. Using single-nucleus transcriptomics, we further revealed that these modules are enriched for specific classes of both excitatory and inhibitory neurons, and immunohistochemistry confirmed expression of highly correlated genes. This unprecedented dataset of patient-specific brain oscillations coupled to genomics unlocks new insights into the genetic mechanisms that support memory encoding.