Molecular and cellular evolution of the primate dorsolateral prefrontal cortex

Abstract

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

Fig. 1.. Transcriptomically-defined cell taxonomy of anthropoid primate dlPFC.

(A) Species (human – red; chimpanzee – blue; macaque – green; marmoset – yellow) and homologous dlPFC areas (blue) analyzed using snRNA-seq. (B) Overview of the snRNA-seq dataset, depicting information from innermost to outermost as: UMAP of all nuclei; dendrogram of hierarchically related subtypes; subtype proportions; marker gene expression; species representation in each subtype (color conforming to panel A); 4 classes; 29 subclasses; 114 refined subtypes. The subtypes detected only in some species are highlighted. See fig. S3A for a more detailed description. IT, intratelencephalic; ET, extratelencephalic; NP, near-projecting; CT, corticothalamic; ChC, chandelier cells; Astro, astrocyte; Micro, microglia; Oligo, oligodendrocytes; OPC: oligodendrocyte precursor cells; Endo, endothelial cells; RB, red blood lineage cells; PC, pericyte; SMC, smooth muscle cells; VLMC, vascular leptomeningeal cell.

Fig. 2.. Shared and divergent features of dlPFC subtypes across anthropoid primates.

(A) Diagram illustrating possible models of species cellular and transcriptomic differences. (B) Subtype abundance comparisons across species. Each pie represents a subtype with size indicating the average subtype proportions across the four species (Materials and Methods). Links represent the expression correlation among subtypes. Subtypes showing species-enrichment in abundance are colored in opaque, otherwise in transparent. Species-specific subtypes are highlighted by dashed circles. (C) Transcriptomic heterogeneity among cells of the same subclasses. Significance was tested via pairwise Wilcoxon Rank Sum test with Bonferroni correction (**: adjusted p < 0.01). (D) Raw transcriptomic divergence across subtypes (columns) in all species pairs (rows). IT, intratelencephalic; ET, extratelencephalic; NP, near-projecting; CT, corticothalamic; ChC, chandelier cells; Astro, astrocyte; Micro, microglia; Oligo, oligodendrocytes; OPC: oligodendrocyte precursor cells; Endo, endothelial cells; RB, red blood lineage cells; PC, pericyte; SMC, smooth muscle cells; VLMC, vascular leptomeningeal cell. huMicro, human-specific microglia (Micro P2RY12 CCL3); hoMicro, Hominini-specific microglia (Micro P2RY12 GLDN). rAstro: reactive astrocyte (Astro AQP4 OSMR).

Fig. 3.. Taxonomy and divergent features of dlPFC excitatory neuron subtypes.

(A) UMAP visualization of all glutamatergic excitatory neuron subtypes with subclasses labeled. (B) Top: UMAP showing all L6 IT-2 subtypes. Bottom: Expression of subtype markers shared in Catarrhini (≥ 2 species) or specific to marmosets. (C) Top: Enrichment of Gene Ontology (GO) terms in the species-enriched genes from each species, with colors indicating the species. Only the GO terms enriched in at least three species are displayed. Bottom: Expression of the selected species-enriched genes involved in the calcium ion binding and extracellular matrix (ECM). ER, endoplasmic reticulum. (D) Expression of genes with high subclass and human specificity. IT, intratelencephalic; ET, extratelencephalic; NP, near-projecting; CT, corticothalamic.

Fig. 4.. Taxonomy and divergent features of dlPFC inhibitory neuron subtypes.

(A) UMAP layout of all GABAergic inhibitory neuron subtypes with subclasses labeled. ChC, chandelier cells. (B) Left: Sankey plots showing correspondence between predicted laminar organization and subtypes/subclasses. Right: SST expression across putative cortical layers. Significance was tested via one-sided Wilcoxon Rank Sum test (***: p < 0.001; ns: not significant). (C) colocalization of SST and RELN (arrowhead) in human dlPFC L1 revealed by double RNA in situ hybridization. Scale bar = 20 μm. (D) Left: log-normalized expression of TH. Middle: Double immunostaining for SST and TH. Right: Proportion of SST+ and SST− cells within TH+ neurons. Scale bars: 20μm in all species. (E) Immunofluorescence staining for TH and SST (left) or DDC (right), combined with SST RNA in situ hybridization. Scale bars: 20μm. (F) Proportion of SST+ and SST− cells in human TH+ neurons.

Fig. 5.. Taxonomy and divergent features of dlPFC glia cell and non-neural cell subtypes.

(A) UMAP showing all glia cell and non-neural cell subtypes in the four primates with subclasses labeled. (B) Standardized average expression of genes (rows) which are species-enriched (top) or -depleted (bottom) across subtypes (columns). Subtypes are color-barcoded on the top with color scheme conforming to panel A. Genes were colored according to the subtypes where they show enrichment or depletion. (C) Transcriptomic divergence (top) and gene expression differences (bottom) of subtypes in the oligodendrocyte lineage between each pair of species. HS: human; PT: chimpanzee; MM: macaque; CJ: marmoset. (D) Left: the two gene modules differentially expressed among the three interlaminar astrocyte subclusters. Right: Expression of the selected genes across the interlaminar and protoplasmic astrocyte subclusters. (E) Top: UMAP plots illustrating microglia and immune subtypes detected in the four primates. Bottom: expression of manually selected subtype markers genes. Marker expression labeling species-specific subtypes are highlighted by dashed rectangles. (F) Left: Illustration of the brain vascular architecture from arteries (red), arterioles, capillaries to veins (blue). Right: Vascular subtypes divided into endothelial cells (left) and mural cells (pericytes and smooth muscle cells, right) located along the arterial-venous axis. Dot plots show expression of marker genes and FLT1 signaling genes. Astro, astrocyte; Micro, microglia; Oligo, oligodendrocytes; OPC: oligodendrocyte precursor cells; Endo, endothelial cells; PC, pericyte; SMC, smooth muscle cells; VLMC, vascular leptomeningeal cell. huMicro, human-specific microglia subtype (Micro P2RY12 CCL3); hoMicro, Hominini-specific microglia subtype (Micro P2RY12 GLDN).

Fig. 6.. Species- and cell type-specific expression of FOXP2.

(A) Violin plots of FOXP2 expression across all subclasses in the four primates and mouse neocortex (17). (B) Immunofluorescent staining against IBA1 (red) combined with RNA in situ hybridization for FOXP2 RNA (green) in L6 of human dlPFC. Arrowheads indicate FOXP2+/IBA1+ microglia, whereas the arrow indicates FOXP2−/IBA1+ microglia. The pie chart summarizes the proportion of FOXP2-expressing cells among IBA1-immunopositive cells. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. (C) Representative images of FOXP2 immunohistochemistry throughout cortical columns in human, chimpanzee, mouse, and echidna. The position of layers and white matter (WM) are indicated. Insets for echidna highlight numerous FOXP2-immunopositive nuclei in the striatum and scarce FOXP2-immunopositive nuclei in the deep neocortical layers. Scale bars: 100 μm for all species. (D) Phylogeny dendrogram of the 51 mammals and their corresponding laminar distribution of FOXP2-immunopositive nuclei.