SUMMARY The objective of this proposal is to elucidate how folic acid prevents neural tube defects on the cellular and molecular level, and to develop a platform to functionally validate and test the FA responsiveness of human NTD candidate genes. Neural tube defects (NTDs) are the second most common congenital abnormality, affecting 6 in 10,000 pregnancies in the United States. Failure to close the neural tube, the precursor to the brain and spinal cord, is complex disorder resulting from genetic and environmental stressors. Because multiple factors play into the development of this disorder, it is critical to understand how environmental factors interact with genetics in the etiology of NTDs. While population-wide folic acid (FA) fortification has reduced the overall NTD rate by 28% in the US, it remains unclear how folic acid prevents NTDs. My proposal aims to address two hypotheses about folic acid and neural tube defects. First, I hypothesize that FA contributes to NTD prevention by acting at the cell movement level as well as inducing transcriptional cascades to compensate for the dysregulated genes. To uncover whether FA can rescue NT closure by acting at the cellular or molecular level, I will characterize the cellular movements and transcriptional profiles for the Alx1 mutant mouse model supplemented with FA. The Alx1 knockout model results in NTDs in 100% of mutant embryos, however prenatal FA supplementation can reduce the incidence to 13%. The Alx1 mutant is a tractable model to dissect the contribution of FA fortification to restoring NT closure because NTDs in this model result from dysregulation of a transcription factor and abnormal cellular movements. We will use imaging-based techniques to characterize cell morphogenesis processes and scRNA-Seq and scATAC-Seq to identify FA-induced gene expression in the Alx1-null background. Results from these complimentary studies will inform if FA acts by supporting cellular movements, inducing transcriptional activity, or a combination. Second, I hypothesize that testing more gene mutations for FA responsiveness will reveal patterns of gene types that benefit from FA fortification as a prevention strategy. To address this hypothesis, I will develop a course-based undergraduate research experience (CURE) entitled The What, the Why, and the How of neural tube defects. The scientific aim of this course will be to functionally validate candidate human NTD genes of unknown significance in NT closure in chick embryos and test the folic acid responsiveness. The societal aim of the CURE is to increase the number of relevant research opportunities for undergraduates while meeting a public health need to screen candidate genes and communicate back to clinicians and families. I expect screening more candidate genes for NTDs and FA responsiveness will reveal new high-risk genes and increase our predictive power over the types of dysregulated genes likely responsive to FA supplementation. Together, the r...