While human cognitive abilities are clearly unique, underlying changes in brain organization and function remain unresolved. Here we characterized the transcriptome of the cortical layers and adjacent white matter in the prefrontal cortexes of humans, chimpanzees and rhesus macaques using unsupervised sectioning followed by RNA sequencing. More than 20% of detected genes were expressed predominantly in one layer, yielding 2,320 human layer markers. While the bulk of the layer markers were conserved among species, 376 switched their expression to another layer in humans. By contrast, only 133 of such changes were detected in the chimpanzee brain, suggesting acceleration of cortical reorganization on the human evolutionary lineage. Immunohistochemistry experiments further showed that human-specific expression changes were not limited to neurons but affected a broad spectrum of cortical cell types. Thus, despite apparent histological conservation, human neocortical organization has undergone substantial changes affecting more than 5% of its transcriptome.
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We thank the Maryland Brain Collection Center and the Chinese Brain Bank Center for providing the human samples; the Biomedical Primate Research Centre, the Burgers' Zoo in Arnhem, and R. Martin and W. Scheffran (Zurich University, Anthropology Department, Zurich, Switzerland) for providing the chimpanzee samples; and the Suzhou Drug Safety Evaluation and Research Center and C. Lian, H. Cai and X. Zheng for providing the macaque samples. We thank G.L. Banes for his comments on the manuscript. This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB13010200 to P.K.); the National Natural Science Foundation of China (grants 91331203, 31171232, 31501047 and 31420103920 to P.K.); the National One Thousand Foreign Experts Plan (grant WQ20123100078 to P.K.); the Bureau of International Cooperation, Chinese Academy of Sciences (grant GJHZ201313 to P.K.); and the Russian Science Foundation (grant 16-14-00220 to P.K.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 The cortical layer assignment to cortical sections in DS2.
For humans or macaques, marked by the silhouette figures, each pair of columns represents a PFC sample from DS2. The leftmost pair represents an average macaque cortical sample based on the three samples of two individuals. The rows represent tissue sections obtained using unsupervised sectioning; the gray scale bar shows section labels. The darker shades of gray represent deeper cortical sections. The colors represent cortical layer assignment based on relative expression of known MLMs across sections. The WM was assigned to sections based on relative expression of the human white matter markers.
Supplementary Figure 2 Correlation distributions based on layer-marker expressions in different datasets and species.
(a) The Pearson’s correlation coefficient (PCC) distribution based on the novel MLMs expression in DS1 and DS2 samples (left), and in DS1 samples and the original LCM macaque samples (right). (b) The PCC distribution based on the HLMs expression in DS1 and DS2 human samples (left), in DS1 human and chimpanzee samples (middle), and in DS1 human and macaque samples (right). The curves show the cumulative frequency of PCC, with the histogram showing the density distribution. The shaded areas show the 95% confidence interval of the cumulative frequency distribution based on 1,000 permutations of the layers. The dash lines show PCC=0.8 threshold and the median PCC for the novel layer markers in each comparison.
Supplementary Figure 3 Gene expression conservation across cortical layers among three species.
(a) The number of genes with section-related expression in each species based on DS1 (humans – red, chimpanzees – green, macaques – blue). (b) The PCC distribution based on the gene expression of section-related genes between each pair of species in DS1: brown – human and chimpanzee, green – chimpanzee and macaque, purple – human and macaque. The section-related genes were defined as genes with section-related expression in at least one species (upper panel), or genes with section-related expression in all three species (lower panel). (c) The PCC distribution based on the gene expression in DS1 human samples and the human ISH data. The gray bars show the chance PCC distribution estimated by permutation of gene labels 1,000 times. (d) Expression of macaque layer markers (MLMs) in human and chimpanzee cortical sections. The MLMs include known MLMs identified based on LCM samples of individual cortical layers (L2-L6) and the novel MLMs. The columns show the average relative expression levels of MLMs specific to a given layer in humans or chimpanzees, as marked by the silhouette figures. The darker shades of blue represent higher relative expression across sections. The gray scale bar shows section labels. The darker shades of gray represent deeper cortical sections. The black boxes and white labels show the cortical sections assigned to the cortical layers marked by the layer markers and white matter markers. The median expression fold change (the median difference between maximum and average expression levels in depicted laminar sections) and its 5%-95% range are shown on top of the heatmap panels.
Supplementary Figure 4 Expression of nine commonly used human and mouse layer markers and one human white matter layer in DS1.
Each triplet of columns shows expression of one layer marker in the three primate species, as marked by the silhouette figures. The darker shades of purple represent higher relative expression across sections. The gray scale bars show section labels. The darker shades of gray represent deeper cortical sections. The black boxes and white labels show the cortical sections assigned to the cortical layers marked by the corresponding commonly used layer marker. The human marker names marked in red. The expression fold change (the difference between maximum and average expression levels in depicted laminar sections) is shown above each column.
Supplementary Figure 5 Layer-specificity of layer markers.
(a) The layer specificities of known MLMs for each layer in L2-6 in each of the three species in DS1. (b) The layer specificity of layer markers identified in DS1. Boxes show distribution of Pearson’s correlation coefficient between the markers’ laminar profiles and the artificial patterns representing each layer. The boxes show the medians and the upper and lower quartiles, the whiskers reach the maximum and minimum points of the distributions. The number of layer markers represented in each panel is shown in the panel. Each row of panels, as shown by the silhouettes, shows the markers found in each of the three species. Each column of panels, on the other hand, shows the distribution for markers of each of the six cortical layers. (c) Layer specificity scores of layer markers identified in DS1. The black shapes show the distribution of the scores, with horizontal lines indicating the mean. Grey shadows show specificity scores below 0.1. The three panels show distributions for markers in macaques (left), humans (center) and chimpanzees (right) as shown in the silhouettes. Numbers of layer markers represented in shapes of each panel are shown on top of the panel.
Supplementary Figure 6 The excess of human-specific changes was robust to sample quality and statistical criteria identifying DE genes between humans and chimpanzees.
(a) RNA sample quality represented as the ratio of transcripts’ 5’ and 3’ coverage. Each box represents the distribution of sample qualities across sections for one individual (n=17 [M1, M2 and M3] or 18 cortical sections). The colors represent species: red – humans, green – chimpanzees, blue – rhesus macaques. The boxes show the medians and the upper and lower quartiles, the whiskers show the extensive range defined as 1.5-fold the range between the first and the third quartiles, the dots show the data points outside the extensive range. (b) The numbers of genes showing each type of significant expression changes on the three evolutionary lineages: human (red), chimpanzee (green) and the lineage connecting the common ancestor of humans and chimpanzees with macaques (blue), estimated based on two human samples (H1 and H3) and two chimpanzee samples (C1 and C3) with comparable sample quality. (c) The number of genes with human or chimpanzee specific changes, with more stringent definition of expression differences between humans and chimpanzees. The heights of bars show the numbers of genes in each category of changes. Dark bars show the numbers of human-specific changed genes and light bars show the numbers of chimpanzee-specific changed genes.
Supplementary Figure 7 Evolutionary properties of T3 expression changes.
(a) The evolutionary rate estimated as the number of genes showing T3 change on the corresponding lineage divided by the lineage length for each of the cortical layers and the underlying WM (human – red, chimpanzee – green, macaque – blue, n=64 jackknife sampling). (b) The relative expression levels of human neuron markers across cortical layers and WM (n=10 genes). (c) The log2-transformed human-chimpanzee ratios based on the numbers of genes assigned to the human and chimpanzee lineages in each of the cortical layers and WM (n=64 jackknife sampling). The variation of the ratio estimates was calculated using jackknife sampling of cortical samples. The boxes show the medians and the upper and lower quartiles, the whiskers show the extensive range defined as 1.5-fold the range between the first and the third quartiles, the dots show the data points outside the extensive range.
Supplementary Figure 8 Numbers of expression-specificity transitions between each pair of cortical layers or WM.
(a) Numbers of expression specificity transitions assigned to the chimpanzee lineage (left) or rhesus macaque / great ape lineage (right), without any fold change cutoff for the laminar expression difference amplitude. (b) Numbers of expression specificity transitions assigned to the human lineage (left), chimpanzee lineage (middle) or rhesus macaque / great ape lineage (right), with two-fold cutoff for the laminar expression difference amplitude.
Supplementary Figure 9 Human-specific L1–WM transitions in DS1 and LCM data.
(a) Spearman’s correlation coefficient of WM/L1 expression fold change between DS1 and human LCM data, across the 60 genes with human-specific L1-WM transition. The red vertical line indicates the estimated correlation (σ=0.344), with the grey curve showing the distribution of Spearman correlation based on 1,000 times of permutation of gene correspondence in the two data sets. (b) Examples of genes showing human-specific L1-WM transition (LPPR1, SERPINB1, EBF4) based on DS1 and LCM-based data. Each symbol represents expression level in one individual in the corresponding tissue. The colors represent the species: red – humans, green – chimpanzees, and blue – rhesus macaques. For each panel, the y-axis shows the expression levels, represented as RPKM, in DS1 (left) or LCM (right).
Supplementary Figure 10 Genes showing human-specific expression-specificity transition from L5 to L3.
(a) Genes with L5-to-L3 transition from mouse to human brains identified using human and mouse brain in situ hybridization. The darker shades of purple represent higher relative expression across sections. The gray scale bars show section labels. The darker shades of gray represent deeper cortical sections. The expression fold change (the difference between maximum and average expression levels in depicted laminar sections) is shown above each column. (b) The 18 genes with human-specific expression specificity transition from L5 to L3. Each row shows the Ensembl ID and gene symbol, as well as the comparison of its expression layer specificity in mice between L3 and L5 (based on data from T.G. Belgard, et al., Neuron 71, 605–616; 2011). Genes with higher expression in L5 than L3 in the mouse dorsal cortex are marked in red, and genes with lower expression in L5 than in L3 are marked in blue. Genes with no mouse orthologous in Ensembl are marked in gray. (c) Expression of one example gene: CLCN6 in DS1 (upper panel) and the mouse dorsal cortex cortical layer samples (T.G. Belgard, et al., Neuron 71, 605–616; 2011) (lower panel).
Supplementary Figure 11 Cell-type-specificity of genes with human-specific changes.
(a) The cell type specificity of the selected genes assessed using immunohistochemistry (IHC), and reported in Y. Zhang, et al., J. Neurosci. 34, 11929–11947; 2014 and M. Uhlen, et al., Science 347, 1260419; 2015. (b) The cell type specificity by IHC experiments of the remaining seven selected genes showing human-specific changes: CLCN6, CNTNAP4, SPEF1, CHRNB3, NME5, NGB and TMEM100. The columns show the average relative expression levels of the corresponding gene in the cortical sections of humans, chimpanzees and macaques, as marked by the silhouette figures. The darker shades of blue represent higher relative expression across sections. The gray scale bar shows section labels. The darker shades of gray represent deeper cortical sections. Each quadruplet of images shows the signal of one gene with neuron marker MAP2 (for CLCN6, CNTNAP4, SPEF1 and TMEM100) or astrocyte marker GFAP (for AQP1 and NGB), and general nuclear marker DAPI. (c) Cell type enrichment analysis indicates general enrichment of genes with human-specific T1, T2 and T3 changes in particular cell types, especially non-neuron cell types. Significant enrichments (FDR<10%) are marked by darker orange, with the mild enrichments (uncorrected P<0.1) marked by light orange. The values show the uncorrected P-values.
Supplementary Figure 12 Dissection of brain samples.
(a) Cortical slices from macaque, chimpanzee and human brains, showing the frontal part of the prefrontal cortex. The dash line ellipses show the location in the dorsolateral prefrontal cortex where “cube” samples were dissected from. Dorsolateral prefrontal cortex was identified as the frontal area of the superior frontal gyrus (SFG), dorsally to the principal sulcus (ps) in macaques, or the superior frontal sulcus (sfs) in chimpanzees and humans. (b) The Nissl staining images of the laminar columns in each of the three species, as shown by the silhouettes. (c) The average thickness of processed cortical cubes for the three species and the relative thickness of the dissected cortical sections by unsupervised sectioning and each layer (G. Rajkowska & P.S. Goldman-Rakic, Cereb Cortex 5, 307–322; 1995 and K. Semendeferi, E. Armstrong, A. Schleicher, K. Zilles & G.W. Van Hoesen, Am J Phys Anthropol 114, 224–241; 2001).
Supplementary Text and Figures
Supplementary Figures 1–12 (PDF 2216 kb)
Supplementary Table 1
DS1 and DS2 sample information. (XLSX 62 kb)
Supplementary Table 2
Gene expression characteristics of the detected genes in DS1, including laminar section-related expression, layer specificity, and lineage-specific changes. (XLSX 3516 kb)
Supplementary Table 3
Primer sequences used in the real-time qPCR; general information on the antibodies used in immunohistochemistry. (XLSX 11 kb)
Supplementary Software (ZIP 4 kb)
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He, Z., Han, D., Efimova, O. et al. Comprehensive transcriptome analysis of neocortical layers in humans, chimpanzees and macaques. Nat Neurosci 20, 886–895 (2017). https://doi.org/10.1038/nn.4548
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