a–c, Heat maps with inferred cell-type homologies highlighted in blue boxes. For each pair of clusters, the shade of grey indicates the minimum proportion of samples that co-cluster. Homologies for human and mouse inhibitory neurons (a), excitatory neurons (b) and non-neuronal cells (c) were predicted on the basis of shared cluster membership using mouse cells from two cortical areas (V1 and ALM) and two unsupervised alignment algorithms (scAlign and Seurat). d, Mouse V1 and mouse ALM excitatory neurons were aligned with scAlign. Blue boxes indicate V1 and ALM clusters that align to the same human clusters in b and are members of homologous cell types. Note that cell types can be matched at higher resolution within species than between species, as expected. e, Left to right: violin plots (n = 10,525 nuclei) showing expression of specific markers of the putative extratelencephalic EXC L4–5 FEZF2 SCN4B cluster (black box) and NPTX1, a gene expressed by all non-PT excitatory neurons. Each row represents a gene, the black dots in each violin represent median gene expression within clusters, and the maximum expression value for each gene is shown on the right-hand side of each row. Expression values are shown on a linear scale. Representative inverted DAPI-stained cortical column (scale bar, 200 µm) with red dots marking the position of cells positive for the genes SLC17A7 and FAM84B and negative for NPTX1 illustrates the relative abundance of the EXC L4–5 FEZF2 SCN4B type in human MTG. Representative examples (arrows) of FAM84B (scale bar, 25 µm) and POU3F1-expressing cells (scale bar, 25 µm). Expression of Fam84b in mouse TEa (scale bar, 75 µm) is shown in the adjacent panel. f, mFISH for NPTX1, a marker of non-PT excitatory types and SLC17A7, shows that NPTX1 labels most SLC17A7+ cells across all cortical layers. Boxed region shown at higher the magnification to the right. One SLC17A7+ cell (white arrow) is NPTX1−, but all other all other SLC17A7+ cells are NPTX1+. Scale bars, 200 µm (left); 50 µm (right). Right, representative inverted DAPI-stained cortical column with red dots that represent SLC17A7+, NPTX1− and POU3F1+ cells. Scale bar, 200 µm. e, f, Experiments were repeated on three donors (human) and two mice with similar results. g, ISH validation of layer distributions in human MTG and mouse primary visual cortex (data from ref. 22). Cells are labelled by cluster marker genes in human (RORB+/CNR1−/PRSS12+) and mouse (Scnn1a+/Hsd11b1+). ISH was performed on three human donors with similar results. For mouse, one experiment was performed.