Pregestational maternal diabetes induces congenital heart defects (CHDs), the most common type of structural birth defects. Although there have been major breakthrough in understanding the genetic causes of these malformations, epidemiological evidence suggests that noninherited factors contribute substantially to the induction of CHDs. Knowledge of how diabetes adversely affects heart development is limited. We found that both maternal type 1 and type 2 diabetes induce oxidative stress and high rates of CHDs, increases apoptosis, decreases cell proliferation, and suppresses Wnt signaling essential for cardiogenesis. Therefore, we hypothesize that maternal diabetes induces oxidative stress in the embryonic heart by increasing NADPH oxidase 4 expression and mitochondrial ROS production. Impaired Wnt signaling mediates the teratogenic effect of diabetes by downregulating Wnt target genes essential for heart development and suppressing key signaling pathways and gene expression in the second heart field progenitors, cardiomyocytes and cardiac neural crest cells leading to CHDs. To test this hypothesis, we propose the following Aims. Aim 1 will determine whether Nox4 is involved in maternal diabetes-induced oxidative stress and abnormal heart formation. We will examine whether diabetes increases Nox4 expression though JNK1/2 and whether Nox4 deletion in the second heart field (SHF) Isl1+ progenitors or cardiac neural crest cells (CNCCs) reduces the teratogenicity of diabetes by inhibiting oxidative stress. Aim 2 will determine whether enhanced mitochondrial ROS production contributes to oxidative stress and abnormal heart formation. We will investigate whether diabetes induces mitochondrial ROS and whether diabetes-increased Nox4 activities contribute to mitochondrial dysfunction and overexpression of the mitochondria-specific antioxidant enzyme SOD2 will reduce the ROS-mediated CHDs. Aim 3 will determine the role of the canonical Wnt antagonist Dkk1 in maternal diabetes-induced heart defects. We hypothesize that deleting Dkk1 reduces CHDs. Aim 4 will investigate the role of the noncanonical Wnt signaling inhibitor, CaMKIIδ, in maternal diabetes- induced heart defects. We propose that diabetes activates CaMKIIδ leading to impaired noncanonical Wnt signaling and heart defects.