Fig. 4: Single-cell transcriptomics reveal subtypes of CD45⁺ cells and their interactions with endothelial cells in prenatal human brain.

a, UMAP plot highlighting 11 distinct CD45⁺ cell subtypes. b, A heat map of marker gene expressions that define each subtype of CD45⁺ cells. MG, microglia; WM, white matter. c, A distribution plot comparing the relative abundance of each CD45⁺ subtype in GE versus CTX. d, GSEA analysis of the bulk RNA-seq data reveal GO terms defined by genes enriched in GE versus CTX. e, A volcano plot showing DEGs identified by pseudobulked scRNA-seq data in GE versus CTX and the GO terms they define. Adjusted P values and fold changes were calculated using DESeq2. By default in DESeq2, the P values attained by the Wald test are corrected for multiple testing using the Benjamini–Hochberg method. The genes shown were filtered to be below the adjusted P value of 0.05 and above a fold change of 1.2 between CTX and GE comparisons (highlighted by the dashed lines). The data in a–c and e are from five independent biological samples at GW17–23. The data in d are from 21 independent biological samples at GW15–23. f, Confocal images from GE and CTX of GW19 and GW23 human brain validating the presence of HLA⁺ cells in GE (yellow arrowheads) but not in CTX. White lines in ‘GE’ panels indicate the ventricular surface, whereas white lines in ‘Cortex’ panels indicate the pia surface. g, Confocal images of VAM markers SCL2A1 and CLDN5 (RNAscope probes) in IBA1⁺ cells in GE (yellow arrowheads) but not in CTX of GW17 and GW21 human brains. White lines indicate the section planes for the orthogonal views of CLND5⁺; IBA1⁺ vasculature-associated MG (right and bottom panels). h, Confocal images of monocyte markers JAML and LYZ (RNAscope probes) in S100A9⁺ cells in GE (yellow arrowheads) but not in CTX of GW17 and GW21 human brains. i, Quantification of the density of HLA⁺ cells, VAM and monocytes in GE and CTX of prenatal human brain. n.s., not significant. Statistics in i use a two-tailed, unpaired Student’s t-test, and the data represent the mean ± standard error of the mean. n indicates the number of independent biological samples used for quantification.