Zebrafish is the leading fish model system in developmental and neurobiology, whereas Mexican tetra is a powerful system for comparative studies. In this study we analyze the conservation and diversification of teleost brain cell types using the zebrafish ( Danio rerio) and the Mexican tetra ( Astyanax mexicanus) as model systems. Little is known about the fate of these duplicated genes in teleosts, their roles in the vertebrate brain, or their links to cellular diversification. Most duplicated genes lose their functions through deleterious mutations (non-functionalization), but genes that are retained may undergo either sub-functionalization (partitioning of functions or gene expression patterns), or neo-functionalization (gain of novel functions or gene expression patterns). It has been hypothesised that the vast diversification in morphology, physiology, and behaviour observed across teleost species was driven by gene family expansions associated with the teleost-specific WGD 4, 12, 14. This group of nearly 30,000 described ray-finned fish species represents the largest clade within vertebrates and has undergone a taxon-specific whole genome duplication (WGD) 12, 13.
Extension of these approaches to more diverse phylogenies is necessary for understanding the molecular and evolutionary basis of cell type conservation and diversification across the tree of life.Ī powerful model for comparative studies of biological diversification are the teleosts. These studies have identified conserved cell types during vertebrate development 7 and mammalian neurogenesis 8, 9, as well as primate-specific adaptations 10, 11. Recently, cross-species comparisons using single-cell RNA-seq have identified shared and species-specific cell types, as well as mechanisms for neuronal evolution 5, 6. Single-cell sequencing has recently emerged as a powerful tool to study and map the cell types of individual species, and has allowed the identification of hundreds of transcriptionally unique cell types in vertebrate tissues, including the brain 5. Biological novelty may arise as a result of gene expansion 4, but it is unknown how the evolution of gene families influences the diversification of cell types in the brain.
Moreover, it is unclear how cell types diversify during evolution or adaptation to extreme environments. These studies have led to the definition of major neuronal classes and subclasses 2, 3 but it is still unknown how molecularly similar or different brain cell types are between species. Since then, the comparison of cell types has largely relied on morphological criteria and, more recently, data from select marker genes 2. The homology of neuronal cell types was first revealed by Ramón y Cajal, who observed that morphologically similar neurons were present in the brains of many species 1. The single-cell atlases presented here are a powerful resource to explore hypothalamic cell types, and reveal how gene family evolution and the neo- and sub-functionalization of paralogs contribute to cellular diversity. Within species comparisons revealed differences in immune repertoires and transcriptional changes in neuropeptidergic cell types associated with genomic differences between surface- and cave-morphs. Species-specific cell types were enriched for the expression of species-specific genes, and characterized by the neo-functionalization of members of recently expanded or contracted gene families. Expression of terminal effector genes, such as neuropeptides, was more conserved than the expression of their associated transcriptional regulators. Orthologous cell types displayed differential paralogue expression that was generated by sub-functionalization after genome duplication. We found that over 75% of cell types were shared between zebrafish and Mexican tetra, which last shared a common ancestor over 150 million years ago. To examine cell type diversity across and within species, we performed single-cell RNA sequencing of ∼130,000 hypothalamic cells from zebrafish ( Danio rerio) and surface- and cave-morphs of Mexican tetra ( Astyanax mexicanus). Hundreds of cell types form the vertebrate brain, but it is largely unknown how similar these cellular repertoires are between or within species, or how cell type diversity evolves.
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