Abstract Many neurobehavioral diseases affect females and males differently, and emerge at different stages of life, leading to the conclusion that one sex is protected from diseases by inherent sex factors, such as gonadal hormones and sex chromosomes. The sites and timing of these effects on brain development are unknown. The method of high-throughput high-precision whole-brain MRI imaging has the power to detect such changes with great sensitivity, in both animal models and humans. This project will exploit these powerful methods to separate gonadal hormonal and sex chromosome effects in the mouse model “Sex Chromosome Trisomy” (SCT), at 9 different ages of development, to discover where and when these factors cause sex differences in brain development (Aim 1). The SCT model compares mice with different numbers and types of sex chromosomes (XX, XY, XXY, XYY), each genotype present in gonadal males or females. Then, a novel pipeline of analysis will compare mouse and human brain development at many stages and in informative groups (differing by age, sex, hormonal status, and sex chromosome complement) to determine which changes in mouse brain are also found in humans, in specific brain regions related to different diseases (Aim 2). The analysis will point to changes in mouse brain that model human brain development. These studies will also pinpoint new sex-biasing effects of hormones and sex chromosomes at localized brain regions at specific developmental stages in mice, leading to further investigation of cellular and molecular changes caused by sex at those sites (Aim 3). Cellular effects (cell size, number, density of defined cell populations) of sex-biasing factors will be measured using CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue- hYdrogel). Gene pathways responding to hormonal and sex chromosome effects in individual cell types in specific brain regions will be measured using single cell RNA-seq. These studies, combining high- resolution neuroimaging, CLARITY, and single cell sequencing, will provide a foundation of concepts about where in the brain, and when during development, specific cell populations respond to sex factors, as a prelude for hypothesizing which sex differences underlie the protective effects of sex- biased factors in cells.