Single-cell volumetric transcriptomic atlas of the adult zebrafish forebrain reveals teleost homologues of cortical and subcortical structures from terrestrial vertebrates

Zebrafish is extensively used for investigating fundamental principles underlying the assembly and the function of the vertebrate brain, most commonly for the study of sensory-motor computations in larval zebrafish midbrain and hindbrain. Accumulating evidence has shown that juvenile and adult zebrafish can perform cognitively demanding tasks such as spatial learning, working memory and social behaviors. Some of these complex behaviors were attributed to distinct forebrain lesions in teleosts, and a few genetic ablations in zebrafish. However, the link between the zebrafish forebrain and vertebrate cortical evolution is not well understood. Here, we generated the Atlas of Zebrafish Transcriptomic Encephalic Cytoarchitecture (AZTEC), the first single-cell volumetric transcriptomic atlas of a teleost forebrain, consisting of 99 multiplexed genes for >350.000 cells. Spatio-molecular clustering of our data and 3D alignment onto an adult zebrafish brain atlas confirm several previously proposed forebrain nuclei, but also reveal multiple novel substructures that were not previously known. We found multiple excitatory and inhibitory neuron types and non-neuronal cells across the zebrafish forebrain with distinct marker genes. While some of these cell types are dispersed widely, several inhibitory and excitatory neuron classes are organized into spatially distinct forebrain nuclei. Integrating AZTEC with single-cell transcriptomes from zebrafish and other vertebrates revealed several cell types and cortical/subcortical regions that are evolutionarily conserved. Functional imaging reveals that the identified zebrafish forebrain regions exhibit spatio-temporally distinct resting-state activity and functional connectivity.