PROJECT SUMMARY/ABSTRACT There are pronounced sex differences in the incidence and etiology of mental health conditions. However, the developmental mechanisms that give rise to sex differences in disease susceptibility or resilience remain largely unknown. In this application we will explore the developmental origins of brain sex differences. In many vertebrates, including mice, sex-specific neural circuitry develops under the control of estrogen signaling during the first few days of life. Treating neonatal females with estrogen irreversibly masculinizes adult social behavior, neural circuitry, and gene expression. To understand the actions of estrogen on the brain, we recently identified the genomic targets of estrogen receptor alpha (ERα) and revealed that these genes are differentially invoked in the developing and adult brain of both sexes. The goal of this proposal is to determine the molecular and circuit consequences of early life ERα actions, with the long-term intent of connecting individual ERα target genes to discrete sex-variable phenotypes. The posterior division of the bed nucleus of the stria terminalis (BNSTp) is a key node in neural circuits that mediate social behaviors and is larger in males compared to females. We hypothesize that BNSTp sexual dimorphism is specified by parallel epigenetic events during a perinatal critical period: initiation of a persistent male-biased gene expression signature and establishment of sex-specific neuronal connectivity. In Aim 1 we will determine the transcriptional mechanisms that define sex differential gene regulation strategies: persistent early life gene expression in males, and fluctuations in response to estrous hormones in adult females. In Aim 2 we will map the connectivity of the two male-biased cell types we previously identified in the BNSTp, in both adults and across postnatal development. In Aim 3 we will test the causality of perinatal ERα target genes and loci in specifying BNSTp sex differences. Taken together, our findings will reveal how gonadal hormone signaling during early life permanently influences adult gene expression, neuronal connectivity, and ultimately, sex-variable behaviors. This work will provide insight into how a transient event during a critical developmental period can have significant impact on the brain and behavior in adulthood. This critical period permanently affects brain structures and function, suggesting that sex differences in psychiatric disorders, such as autism and depression, may originate during sexual differentiation of the brain.