Molecular and spatial mapping of hypothalamic cell-types using the Xenium analyzer

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PROJECT SUMMARY Steroid hormones in humans and other animals coordinate physiological and behavioral processes underlying optimal responses to the social environment. The brain is a major site of steroid hormone action; however, our knowledge of the role of steroid hormones in regulating gene expression and neuroplasticity in the brain is in its infancy. My research program aims to uncover the connections between steroid hormones, gene expression in the brain, and specific cellular functions using Astatotilapia burtoni, a cichlid fish that exhibits sophisticated social hierarchies. In the wild as in the laboratory, male A. burtoni stratify along a social hierarchy where dominant males possess bright coloration, aggressively defend a territory, and mate with females, while non- dominant males do not. Female A. burtoni do not form a social hierarchy but behave aggressively towards one another for mating opportunities. Social rank is in flux, as dominant and non-dominant males can change their status depending on the social milieu. These complex social interactions are tightly linked to levels of a class of steroid hormones called androgens. My research program leverages the social dynamics of A. burtoni in the laboratory to discover the role of androgens in controlling genes in the brain and neuroplasticity. In our ongoing work, we have generated a cell-type specific molecular atlas of the A. burtoni hypothalamus using single-cell RNA-sequencing (scRNAseq). The hypothalamus is a conserved brain region that coordinates adaptive social behaviors across vertebrates. While scRNAseq provides cell-type resolution of specific gene expression patterns, it does not tell you where the specific cells are anatomically within the region of interest. The current proposal aims to address this issue using the 10X Genomics Xenium in situ analyzer, an end-to-end gene expression analysis platform that can label hundreds of genes simultaneously in fresh-frozen or formalin-fixed, sectioned tissue. Using the Xenium will allow for the spatial resolution of the distinct cell-types we have identified in our current scRNAseq dataset. We will combine genetic markers of all cell-types with candidate genes such as distinct androgen receptor (AR) genes present in our Xenium analysis, revealing precisely which cells and where are AR+, providing a precise road-map for testable hypotheses related to the molecular and cellular control of social behavior. Our hypothalamic spatial transcriptome will be compared to the currently only available hypothalamic spatial transcriptome in mice to reveal novel insights into the genetic signatures of AR+ cell types and other cell populations in two species used heavily in social behavior studies. With the generation of a molecular and spatial map of the A. burtoni hypothalamus, we will be able to address fundamental questions regarding the molecular and neural control of the brain and social behavior. Indeed, these questions may connect naturally to those on the control of social behavior in other species such as humans and how social systems emerge throughout evolution.