Adult sex change leads to extensive forebrain reorganization in clownfish
Sex differences in the brain have been demonstrated in all major vertebrate lineages but are not well understood at a molecular and cellular level. Sex-changing fishes provide a unique opportunity to uncover mechanisms underlying sexual differentiation of the brain and regulation of sexual phenotypes. Sex change requires the complete transformation of the gonads and behaviors, which in turn require transformation of sexually-differentiated control mechanisms in the brain. However, detailed molecular and cellular profiling of sex differences in the brains of sex-changing fish is lacking. In this study we applied single nucleus RNA-sequencing (snRNA-seq) to generate the first atlas of sex differences in preoptic area (POA) and telencephalon of the model anemonefish Amphiprion ocellaris. We uncovered remarkably widespread sex differences in cell-type abundance and cell-type transcriptome.  The most prominent difference was observed in the dorsal medial telencephalon where females displayed more than twice as many glutamatergic neurons as males and more than 400 differentially expressed genes consistent with immature neuronal development in males relative to females.  These results provide an unparalleled level of depth to our understanding of sexual differentiation in the brain of a sex-changing fish, and richly characterizes numerous specific sexual dimorphisms that must develop during sex change. Overall design: Single nuclei RNA sequencing was performed on nuclei isolated by Fluorescence-activated cell sorting (FACS) from the combined telencephalon and POA of Amphiprion ocellaris (common clownfish) males and females for comparison between the sexes (two replicates per sex, three individuals pooled per replicate).