Single-cell genomics of the mouse olfactory cortex reveals contrasts with neocortex and ancestral signatures of cell type evolution

Understanding the molecular logic of cortical cell-type diversity can illuminate cortical circuit function and evolution. Here, we performed single-nucleus transcriptome and chromatin accessibility analyses to compare neurons across three- to six-layered cortical areas of adult mice and across tetrapod species. We found that, in contrast to the six-layered neocortex, glutamatergic neurons of the three-layered mouse olfactory (piriform) cortex displayed continuous rather than discrete variation in transcriptomic profiles. Subsets of piriform and neocortical glutamatergic cells with conserved transcriptomic profiles were distinguished by distinct, area-specific epigenetic states. Furthermore, we identified a prominent population of immature neurons in piriform cortex and observed that, in contrast to the neocortex, piriform cortex exhibited divergence between glutamatergic cells in lab versus wild-derived mice. Finally, we showed that piriform neurons displayed greater transcriptomic similarity to cortical neurons of turtles, lizards, and salamanders than to those of the neocortex. In summary, despite over 200 million years of co-evolution alongside the neocortex, olfactory cortex neurons retain molecular signatures of ancestral cortical identity. Nuclei from micro-dissected cortical areas were isolated in C57Bl/6 mice and wild-derived mice, and 10x multiome or 10x RNA sequencing 3.1 version (only for some wild-derived mice replicates) were performed. Areas collected were: A = anterior piriform cortex P = posterior piriform cortex T = agranular insular cortex N = primary somatosensory cortex W = data from wild-derived mice 1, 2, 3... = biological replicate ID