Adult sex change leads to extensive forebrain reorganization in clownfish

Background: Sexual differentiation of the brain occurs in all major vertebrate lineages but is not well understood at a molecular and cellular level. Unlike most vertebrates, sex-changing fishes have the remarkable ability to change reproductive sex during adulthood in response to social stimuli, offering a unique opportunity to understand mechanisms by which the nervous system can initiate and coordinate sexual differentiation. Methods: This study explores sexual differentiation of the forebrain using single nucleus RNA-sequencing in the anemonefish Amphiprion ocellaris, producing the first cellular atlas of a sex-changing brain. Results: We uncover extensive sex differences in cell type-specific gene expression, relative proportions of cells, baseline neuronal excitation, and predicted inter-neuronal communication. Additionally, we identify the cholecystokinin, galanin, and estrogen systems as central molecular axes of sexual differentiation. Supported by these findings, we propose a model of sexual differentiation in the conserved vertebrate social decision-making network spanning multiple subtypes of neurons and glia, including neuronal subpopulations within the preoptic area that are positioned to regulate gonadal differentiation. Conclusions: This work deepens our understanding of sexual differentiation in the vertebrate brain and defines a rich suite of molecular and cellular pathways that differentiate during adult sex change in anemonefish.