Project Summary/Abstract Alcohol (ethanol) exposure during pregnancy is the leading environmental cause of birth defects and central nervous system dysfunction. While the effects of ethanol on the brain and face have been explored quite extensively, there is a considerable lack of knowledge regarding the pathogenic mechanisms of developmental ethanol exposure. A significant amount of work over the years, including from our lab, has demonstrated that apoptosis is associated with prenatal ethanol exposure. However, apoptosis cannot account for all of the effects associated with fetal alcohol spectrum disorders (FASD). Previous work on this project has demonstrated that one non-apoptotic pathogenic mechanism involves the primary cilia. Primary cilia are small hair-like organelles that are present on almost every cell and are critical for normal development and cellular homeostasis. Genetic disruptions in primary cilia function have been increasingly explored over the last several years and are classified under the term ciliopathies. Recent work from our lab has demonstrated that prenatal ethanol exposure can also temporarily disrupt primary cilia function resulting in a “transient ciliopathy” with a particularly devastating effect on the Sonic hedgehog (Shh) pathway. However, the cellular mechanism by which ethanol acts at the cilium is unclear. In this current proposal, we will further explore these mechanisms using a variety of tools and cross- sectional approaches. In Aim 1, we will test whether ethanol is affecting transport along the microtubule axoneme within the primary cilium or transport into or out of the primary cilia compartment. Both of these ciliary functions are vital to normal cilia function and will be probed using a variety of in vitro and in vivo techniques in wild type and transgenic mouse lines. In Aim 2, we will take advantage of the convergent effects of ethanol and cannabinoids on primary cilia to explore the role of ethanol in modulating G-protein coupled receptor (GPCR) signaling. Several GPCRs, including those in the Shh and cannabinoid pathways signal through primary cilia and this aim will test how ethanol affects these key receptors. Aim 3 will leverage targeted small molecule screens to identify novel pathways of ethanol's pathogenic mechanisms. Using a combination of high-throughput in vitro assays and in vivo confirmation, we will identify novel targets of developmental ethanol exposure. Collectively, these experiments will greatly increase our knowledge of ethanol's mechanisms of actions in the developing embryo, with important implications for informing clinical research studies and prevention and intervention strategies as well.