Variation in neuronal activity state, axonal projection target, and position principally define the transcriptional identity of individual neocortical projection neurons.
Single-cell RNA sequencing technologies have generated the first catalogs of transcriptionally defined neuronal subtypes of the brain. However, the biologically informative cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1023 cellular responses to manipulation of sensory input demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type specific biases in the choice of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype.