Molecular diversity and evolution of neuron types in the amniote brain

The existence of evolutionarily conserved regions in the vertebrate brain is well established. The rules and constraints underlying the evolution of neuron types, however, remain poorly understood. To compare neuron types across brain regions and species, we generated a cell type atlas of the brain of a bearded dragon and compared it with mouse datasets. Conserved classes of neurons could be identified from the expression of hundreds of genes, including homeodomain-type transcription factors and genes involved in connectivity. Within these classes, however, there are both conserved and divergent neuron types, precluding a simple categorization of the brain into ancestral and novel areas. In the thalamus, neuronal diversification correlates with the evolution of the cortex, suggesting that developmental origin and circuit allocation are drivers of neuronal identity and evolution. Description Lizard traces in the mouse brain Although lizards and mammals share common evolutionary roots, their pathways to today diverged hundreds of millions of years ago. Using single-cell transcriptomics, Hain et al. compared the brain of the lizard Pogona vitticeps, known as the Australian dragon, with that of the mouse (see the Perspective by Faltine-Gonzalez and Kebschull). They found that certain neuronal identities, marked by what transcription factors they express, are conserved from lizard to mammal, and others show evidence of evolutionary innovation. Therefore, the mammalian brain is an interwoven tapestry of new and ancestral traces. —PJH The mammalian and reptilian brains both result from parallel diversification processes from common ancestral neuron type families. INTRODUCTION Vertebrate evolution took an important turn before the onset of the Permian, 320 million years ago, with the transition of early tetrapods from water to land, the appearance of amniotes, and, soon thereafter, their bifurcation into sauropsids (future reptiles and birds) and synapsids (future mammals). Despite this branched history, the brains of all tetrapods share the same ancestral architecture defined by brain regions established during embryonic development (pallium, subpallium, thalamus, cerebellum, etc.) and by their long-range connections. Yet how variations on this common organization contributed to lineage- and species-specific adaptations is not clear. A commonly held assumption, for example, is that subcortical regions are ancient and “deeply conserved,” whereas the mammalian cortex is “new,” following profound changes in cortical development in this lineage. Brain regions, however, do not operate in isolation, raising the possibility that the evolution of interconnected neurons might be correlated. Likewise, areas in reptiles and mammals that derive from a common ancestral structure, such as the cerebral cortex, may have evolved in each lineage in such a way that they now each contain both ancient (thus common) and novel neuron types. Because traditional comparisons of developmental regions and projections may not suffice to reveal these similarities and differences, we investigated these issues using cellular transcriptomic approaches. RATIONALE Neurons are the most diverse cell types in the brain; their evolutionary diversification reflects changes in the developmental processes that produce them and, in turn, may drive changes in the neural circuits to which they belong. To the extent that a neuron’s transcriptome represents the molecular encoding of its identity, connectivity, and developmental and evolutionary histories, comparing neuronal transcriptomes across species should yield insights into brain evolution. To elucidate the evolution of neuronal diversity across brain regions, we generated a cell type atlas of the brain of a reptile, the Australian bearded dragon Pogona vitticeps, and compared it with existing mouse brain datasets. RESULTS We profiled 285,483 single-cell transcriptomes from the brain of Pogona and identified and annotated 233 distinct types of neurons. Computational integration of this dataset with publicly available mouse data revealed that lizard and mouse neurons co-cluster according to their regional and neurotransmitter identities. These integrated clusters expressed distinctive combinations of developmental transcription factors (including homeodomain-type) and genes involved in neuronal connectivity (cell junction, synaptic signaling, neuronal projections, synaptic transmission), indicating that both developmental origin and circuit allocation define broad, evolutionarily conserved classes of neurons in the amniote brain. At a finer level, these broad classes included neuron types with a wide range of transcriptomic variation across species. Certain neuron types could be readily mapped from lizard to mouse, indicating high transcriptomic similarity; others, however, could not be mapped across species unambiguously, owing to transcriptomic divergence. This dichotomy was true for all regions analyzed (telencephalon, diencephalon, and midbrain), indicating that neuronal diversification is ubiquitous in these brain regions. This was particularly evident in the thalamus, where neurons with high transcriptomic similarity across species (GABAergic reticular thalamic nucleus, glutamatergic “medial thalamus”) are juxtaposed with neuron types with diverging gene expression (glutamatergic “lateral thalamus”). In the lateral thalamus, lizard and mouse neurons from sensory relay nuclei did not co-cluster according to sensory modality, suggesting that these neurons may have diversified extensively, reflecting the different fates of their cortical partners in the reptilian and mammalian lineages. CONCLUSION Using comparative single-cell transcriptomics, we identified a core set of neuron types with high transcriptomic similarity between the brains of a lizard and a mammal, despite 320 million years of separate evolution. These neuron types are not restricted to subcortical regions but are found everywhere in the brain, including in the cerebral cortex, challenging the notion that certain brain regions are more ancient than others. Our data suggest that, even if the brain consists of developmental modules defined by ancient and shared molecular determinants, the evolution of the brain acts upon each module by keeping (e.g., reticular thalamic nucleus and medial thalamus) or diversifying (e.g., lateral thalamus) neuron types in a manner that is correlated with local and long-range connectivity. Transcriptomic study of neuronal evolution among amniotes. Reptiles and mammals evolved independently of each other for ~300 million years. We generated a cell type atlas from the brain of a lizard, Pogona vitticeps. Computational integration of these data with mouse transcriptomes reveals that telencephalon, diencephalon, and mesencephalon each contain mixtures of similar and divergent neurons, indicating that neuron diversification is ubiquitous in those regions.