Extended Data Fig. 9: Single-cell transcriptomic profiling of FEZF2 and TSHZ3 depletion in cortical chimeroids.

a) Differential gene expression (DEG) analysis comparing the molecular effects of FEZF2 shRNA, TSHZ3 shRNA, and 2x-KD conditions to that of scram shRNA. Double-knockdown had additive effects on both the number of DEGs and the magnitude of gene expression changes relative to scram shRNA-expressing cells (left boxplots), with 2x-KD shRNA affecting the expression of synaptic genes both in deep layer neurons and radial glia. Bar graphs (left) plot the number of differentially expressed genes (DEGs) in each comparison, for each cell type, while boxplots show that the average gene scores of these DEGs are significantly higher in the 2x-KD conditions relative to single knock-down conditions. In deep layer cells, there were 305 DEGs, 421 DEGs, and 469 DEGs for FEZF2-shRNA, TSHZ3-shRNA, and 2x-KD conditions. In radial glia, there were 264 DEGs, 128 DEGs, and 370 DEGs for FEZF2-shRNA, TSHZ3-shRNA, and 2x-KD conditions. Significance calculated with two-sided Welch’s t-tests. Gene ontology terms that are enriched within these DEG sets are displayed in dot plots (left). Individual terms are shown with adjusted p-value represented as dot color and the percent of term-associated genes that are identified as DEGs. b) Emergence of a unique neuronal cell type in 2x-KD conditions. Top right UMAP shows that cells in the cluster 15 (generated using standard Louvain clustering using Seurat) are composed almost entirely of 2x-KD cells (inset), further shown by middle pie chart displaying composition of experimental conditions among cluster 15 cells. Cluster 15 cells are predominantly composed of a deep layer subset (left pie chart), but also contains other excitatory neuronal subtypes. The neuronal identity of this cluster is further supported by gene ontology term enrichment analysis of cluster 15 markers, demonstrating the prevalence of synaptic complexes. Individual terms are shown with adjusted p-value represented as dot color and the percent of term-associated genes that are identified as DEGs. Of note, an analog for cluster 15 cells in the adult meta-atlas was not identified.