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 considerable variation in the consequences of developmental ethanol exposure. Some of this variation is due to differences in timing and amount of exposure or nutritional factors. However, even when controlling for these factors, it is still clear that not everyone exposed to ethanol during development is affected equally and it has become increasingly clear that genetic factors play a very significant role in fetal alcohol spectrum disorders (FASD). Historically, elucidating these genetic factors that mediate risk or resilience has been relatively slow and characterized by human association studies or quantitative trait loci mapping in animal models. More recently, we have applied next generation sequencing technologies and forward genetic screens to more rapidly identify genes and pathways that alter susceptibility to prenatal ethanol exposure. These studies have taken advantage of varied mouse strains with differential alcohol susceptibility, numerous transgenic mouse lines, and high throughput zebrafish genetic analyses and CRISPR/Cas9 gene editing techniques. In this current proposal, we will further these approaches with the combination of the powerful genomic analyses capabilities of the Collaborative Cross mouse genetics tools. In Aim 1, we will explore mechanisms underlying ethanol sensitivity using complementary mouse and fish transgenic lines, while identifying further candidate genes via comparisons of the highly ethanol susceptible mouse strain, C57BL/6J vs. the highly ethanol resistant strain 129S1/Svlmj. This comparison will be aided by embryonic transcriptomic (RNA-Seq) analyses, selective crossbreeding to induce susceptibility in a resistant line (129) with extensive genome sequencing analyses. Aim 2 will significantly expand our genomic analyses by examining CC founder strains for their susceptibility to ethanol followed by transcriptomic profiling of susceptible and resistant strains. Conserved candidate genes and pathways will be further tested and characterized in our high throughput zebrafish phenotyping analyses. Aim 3 will take a complementary bioinformatic approach by identifying chemical modifiers of gene-ethanol interactions in a high throughput zebrafish screen with further confirmation in our mouse model of FASD. This proposal brings together experts on mouse and fish genetics, embryology and alcohol teratology. Together, these experiments will greatly enhance our understanding of the genetic etiology of ethanol-induced brain and craniofacial malformations during early embryonic development, as well as aiding in the identification of the pathogenic mechanisms involved in FASD.