Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization1–5. First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.

The amygdaloid complex consists of diverse nuclei that belong to distinct functional systems, yet many issues about its development are poorly understood. Here, we identify a stream of migrating cells that form specific amygdaloid nuclei in mice. In utero electroporation showed that this caudal amygdaloid stream (CAS) originated in a unique domain at the caudal telencephalic pole that is contiguous with the dorsal pallium, which was previously thought to generate only neocortical cells. The CAS and the neocortex share mechanisms for specification (transcription factors Tbr1, Lhx2 and Emx1/2) and migration (reelin and Cdk5). Reelin, a critical cue for migration in the neocortex, and Cdk5, which is specifically required for migration along radial glia in the neocortex, were both selectively required for the normal migration of the CAS, but not for that of other amygdaloid nuclei. This is first evidence of a dorsal pallial contribution to the amygdala, demonstrating a developmental and mechanistic link between the amygdala and the neocortex.

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels.