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.

This study provides key insights into brain sex differences in sex-changing anemonefish (Amphiprion ocellaris), a species that changes sex in adulthood in response to the social environment. Using single nucleus RNA-sequencing, the study provides the first brain cellular atlas showing sex differences in two crucial reproductive areas: the preoptic area and telencephalon. The research identifies notable sex-differences in cell-type proportions and gene expression, particularly in radial glia and glutamatergic neurons that co-express the neuropeptide cholecystokinin. It also highlights differences in preoptic area neurons likely involved in gonadal regulation. This work deepens our understanding of sexual differentiation of the brain in vertebrates, especially those capable of adult sex change, and illuminates key molecular and cellular beginning and endpoints of the process.

Spatially resolved cell atlas of the teleost telencephalon and deep homology of the vertebrate forebrain.

The telencephalon has undergone remarkable diversification and expansion throughout vertebrate evolution, exhibiting striking variations in structural and functional complexity. Nevertheless, fundamental features are shared across vertebrate taxa, such as the presence of distinct regions including the pallium, subpallium, and olfactory structures. Teleost fishes have a uniquely "everted" telencephalon, which has confounded comparisons of their brain regions to other vertebrates. Here we combine spatial transcriptomics and single nucleus RNA-sequencing to generate a spatially-resolved transcriptional atlas of the Mchenga conophorus cichlid fish telencephalon. We then compare cell-types and anatomical regions in the cichlid telencephalon with those in amphibians, reptiles, birds, and mammals. We uncover striking transcriptional similarities between cell-types in the fish telencephalon and subpallial, hippocampal, and cortical cell-types in tetrapods, and find support for partial eversion of the teleost telencephalon. Ultimately, our work lends new insights into the organization and evolution of conserved cell-types and regions in the vertebrate forebrain.

Adult sex change leads to extensive forebrain reorganization in clownfish.

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. 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. 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 neurosexual 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. 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.

Natural variation in oxytocin receptor signaling causes widespread changes in brain transcription: a link to the natural killer gene complex.

Oxytocin (OXT) is a highly conserved neuropeptide that modulates social cognition, and variation in its receptor gene (Oxtr) is associated with divergent social phenotypes. The cellular mechanisms connecting Oxtr genotype to social phenotype remain obscure. We exploit an association between Oxtr polymorphisms and striatal-specific OXTR density in prairie voles to investigate how OXTR signaling influences the brain transcriptome. We discover widespread, OXTR signaling-dependent transcriptomic changes. Interestingly, OXTR signaling robustly modulates gene expression of C-type lectin-like receptors (CTLRs) in the natural killer gene complex, a genomic region associated with immune function. CTLRs are positioned to control microglial synaptic pruning; a process important for shaping neural circuits. Similar relationships between OXTR RNA and CTLR gene expression were found in human striatum. These data suggest a potential molecular mechanism by which variation in OXTR signaling due to genetic background and/or life-long social experiences, including nurturing/neglect, may affect circuit connectivity and social behavior.

Cellular profiling of a recently-evolved social behavior in cichlid fishes.

Social behaviors are diverse in nature, but it is unclear how conserved genes, brain regions, and cell populations generate this diversity. Here we investigate bower-building, a recently-evolved social behavior in cichlid fishes. We use single nucleus RNA-sequencing in 38 individuals to show signatures of recent behavior in specific neuronal populations, and building-associated rebalancing of neuronal proportions in the putative homolog of the hippocampal formation. Using comparative genomics across 27 species, we trace bower-associated genome evolution to a subpopulation of glia lining the dorsal telencephalon. We show evidence that building-associated neural activity and a departure from quiescence in this glial subpopulation together regulate hippocampal-like neuronal rebalancing. Our work links behavior-associated genomic variation to specific brain cell types and their functions, and suggests a social behavior has evolved through changes in glia.

Spatially resolved cell atlas of the teleost telencephalon and deep homology of the vertebrate forebrain.

The telencephalon has undergone remarkable diversification and expansion throughout vertebrate evolution, exhibiting striking differences in structural and functional complexity. Nevertheless, fundamental features are shared across vertebrate taxa, such as the presence of distinct regions including the pallium, subpallium, and olfactory structures. Teleost fishes have a uniquely 'everted' telencephalon, which has made it challenging to compare brain regions in fish to those in other vertebrates. Here we combine spatial transcriptomics and single-nucleus RNA-sequencing to generate a spatially-resolved transcriptional atlas of the cichlid fish telencephalon. We then compare cell-types and anatomical regions in the cichlid telencephalon with those in amphibians, reptiles, birds, and mammals. We uncover striking transcriptional similarities between cell populations in the fish telencephalon and subpallial, hippocampal, and cortical cell populations in tetrapods. Ultimately, our work lends new insights into the organization and evolution of conserved cell-types and regions in the vertebrate forebrain.