Cell types in macaque embryonic and fetal brain development. (A) Schematic diagram of sample collecting and data analyzing. We collected the parietal lobe from the embryos across developmental stages from E40 to E90. UMAP visualization of snRNA-seq data from individual time points. (B) The transcriptome data of single cells were collected and used to do clustering using Seurat. Visualization of major types of cells using UMAP. Dots, individual cells; color, clusters. (C) Violin plot of molecular markers for annotating cell types. (D) The expressions of the classic marker genes for each cell type were plotted to UMAP visualization. Light grey, no expression; Dark blue, relative expression.

Excitatory neuron subclusters in the developing macaque cerebral cortex. (A) Left, Clustering of excitatory neuron subclusters collected at all time points, visualized via UMAP. Cells are colored according to subcluster identities (left) timepoint of collection(right). (B) Differentially expression of deep-layer marker BCL11B and upper layer marker CUX1 is highlighted, and the combined excitatory neuron populations (all time points). (C) Excitatory neuron subclusters UMAP plot shows the expression of classic markers for deep layers (BCL11B, FEZF2, SOX5) and upper layers (CUX1, SATB2) present each time point. (D) Proportion of different excitatory neuron subtclusters corresponding to excitatory neurons in each time point.

Cell diversity and regulation of progenitor cells in the macaque cortical neurogenesis. (A) UMAP shows eight progenitor clusters and cell annotation. Left, cells are colored according to seurat clusters; right, cells are colored according to the collection time point. (B) Feature plot of outer radial glia marker genes HOPX shows higher expression in C10-C14-C12 (left). Feature plots of Intermediate progenitor cell marker gene EOMES show higher expression in C10-C22-C8 (right). (C) Pseudotime analysis by Slingshot of HOPX-positive cells (C10-C14-C12). The Slingshot result with the lines indicating the trajectories of lineages and the arrows indicating directions of the pseudotime. Cells are colored according to cell (C) and pesudotime (D). Dots: single cells; colors: cluster and subcluster identity. (E) Heatmap shows the relative expression of top150 genes displaying significant changes along the pseudotime axis of RG to oRG (C10-C14-C12). The columns represent the cells being ordered along the pseudotime axis.

Transcriptional regulation of excitatory neuron lineage during embryonic cortical neurogenesis. (A) UMAP shows the alignment of macaque cortical NPCs, IPCs, and excitatory neurons. Left, cells are colored according to cell annotation; right, cells are colored according to the time point of collection. (B) Dot plot showing the marker genes for the deep layer excitatory neuron (FEZF2) and upper-layer excitatory neuron (DOK5). Light grey, no expression; Dark blue, relative expression. (C) Pseudotime analysis by Slingshot projected on PCA plot of RGCs, oRGCs, IPCs, and excitatory neuron subclusters. The Slingshot result with the lines indicating the trajectories of lineages and the arrows indicating directions of the pseudotime. Dots: single cells; colors: cluster and subcluster identity. Framed numbers marked start point, endpoint, and essential nodes of Slingshot inference trajectory. Framed numbers”1” was excitatory neuron lineage trajectory start point (C10). Framed number”4” marked immature neurons. Framed numbers “2” and “3” marked deep-layer neurons and upper-layer neurons. Cells are colored according to cell annotation and pseudotime. (D) Heatmap shows the relative expression of top100 genes displaying significant changes along the pseudotime axis of each lineage branch. The columns represent the cells being ordered along the pseudotime axis. (E) Left, Slingshot branching tree related to Slingshot pseudotime analysis in C. The root is E40 earliest RG (C10), tips are deep layer excitatory neurons generated at the early stage (E40, E50), and upper-layer excitatory neurons generated at the later stage (E70, E80, E90). Right, Branching trees showing the expression of marker genes of apical progenitors (PAX6), outer radial glia cells (HOPX), intermediate progenitors (EOMES), and excitatory neurons (NEUROD2), as well as genes in Figure 3E, including callosal neurons (SATB2, CUX2), deeper layer neurons (SOX5, FEZF2), corticofugal neurons (FEZF2, TLE4). There is a sequential progression of radial glia cells, intermediate progenitors, and excitatory neurons.

Integration of human, macaque and mouse single-cell dataset reveals conserved and divergent progenitor cell types. (A) Left: UMAP plot of cross-species integrated single cell transcriptome data with liger. Colors represent different major cell types (Black: Human dataset; Dark grey: macaque dataset; Liger grey: mouse). Right: The UMAP plot of each dataset, colored by ligercluster. (B) The expressions of the classic oRG marker genes were plotted to UMAP visualization. Light grey, no expression; Dark blue, relative expression. (C) Comparison of vRG→oRG and vRG→IPC developmental trajectories between human, macaque, and mouse.

The cell-intrinsic patterns of transcriptional regulation comparative analysis responsible for generation of vRGs. (A) Normalized gene expressions of human, macaque and mouse are distributed within five sequential AP transcriptional states. (B) Temporal expression heatmap of homologous transcription factor genes of among human, macaque, and mouse temporal expression heatmap. (The TFs genes in the dashed boxes showed similar temporal expression patterns across species. (C) and (F) Top 7 regulon specificity score gene at macaque and mouse each embryonic cortex development timepoint. (D) and (G) Gene-expression heatmap of top 7 regulon specificity score gene in (C) and (F) Color scale: red, high expression; blue, low expression. (E) and (H) Protein-protein interactions analysis of macaque and mouse potential transcriptional regulation in the macaque and mouse cortex neurogenesis using the STRING database (http://string-db.org). Nodes are the top 7 regulon specificity score TFs genes at each embryonic development timepoint and their top 5 target genes analysis by SCENIC. Node size is positively correlated with the number of directed edges.

Sample collection and Quality Control.

(A) Schematic diagram of sample collecting anatomical area.

(B)The cell number per sample after quality control.

(C) Single-cell transcriptome library information for each sample.

scRNA-Seq uncovers cell type in the developing macaque neocortex.

(A) Visualization of different dimensionality reduction of all cells. (Left, UMAP visualization with UMAP1 and UMAP2; right, 3D model of UMAP visualization with UMAP1, UMAP2, and UMAP3).

(B) Top marker genes for each of the 28 cell clusters shown in Figure 1B.

(C) Feature plots of marker gene expression. Colors represent scaled gene expression.

Additional information for excitatory neuron subcluster.

(A) UMAP visualization of excitatory neuron subcluster cell scRNA-seq data from individual time points. Cells are colored by excitatory neuron subcluster assignment.

(B) Top marker genes for each of the excitatory neuron subclusters.

Stem and Excitatory neuron subclusters mapping genes.

Developmental regulation of gene expression from RG to IPC.

(A) Pseudotime analysis by Slingshot of EOMES-positive cells (C10-C22-C8). Dots: single cells; Cells are colored by their identity.

(B) Heatmap shows the relative expression of top150 genes displaying significant changes along the pseudotime axis of RG to IPC (C10-C22-C8). The columns represent the cells being ordered along the pseudotime axis. Start point, endpoint, and important nodes of Slingshot inference trajectory were marked by framed numbers. Framed numbers”2” were radial glia cells at the beginning of pseudo time (C10). Framed numbers”1” and “3” marked intermediate state nodes. Framed number “4” marked intermediate progenitor cell (C8).

Transcriptional regulation of OPC differentiation into astrocytes and oligodendrocytes.

(A) Feature plot of classic marker for astrocytes (AQP4) and oligodendrocytes (OLIG2).

(B) Slingshot branching tree related to Slingshot pseudotime analysis in (C) and (D). Root is OPC (C16), tips astrocytes (C13), and oligodendrocytes (C18).

(C and D) Pseudotime analysis by Slingshot of glial cell lineage (C16→C13, C16→C12). The Slingshot result with the lines indicating the trajectories of lineages and the arrows indicating directions of the pseudotime. Cells are colored according to their identity (C) and pesudotime (D). Dots: single cells; colors: cluster and subcluster identity.

(E) Heatmap shows the relative expression of top150 genes displaying significant changes along the pseudotime axis of glial cell lineage (C16→C13, C16→C12). The columns represent the cells being ordered along the pseudotime axis.

Upper layer and deep layer excitatory neuron proportion analysis among species

(A) Cell type annotation of integrated dataset.

(B and C) Feartureplot shows the express of upper layer marker genes CUX2, POU3F2, SATB2 and deep layer marker genes FEZF2, SOX5.

(A) Proportion analysis of excitatory neuron subclusters in human, macaque, and mouse datasets at different developmental timepoint.

Temporal expression pattern of RNA binding protein and transcription factor genes in human, macaque, and mouse vRG.

(A) Temporal expression heatmap of RNA binding protein genes in human, macaque, and mouse ventricular radial glia.

(B and C) Temporal expression heatmap of Transcription factors genes in human, macaque and mouse ventricular radial glia sorted by temporal pattern of human (B) and mouse (C). (Note: each line is the same homologous gene).