Overburdened Ferroptotic Stress Impairs Tooth Morphogenesis

Abstract Regulated cell death (RCD) has been reported to play a critical role in organ development. Despite apoptosis, the best known and most studied RCD in organogenesis, whether newly identified non-apoptotic cell death participates in the physiological process like development is barely understood. As a metabolic-related cell death pathway, ferroptosis, characterized by iron-dependent and lethal lipid peroxidation, was firstly described in 2012 and is now being wildly explored for its pathological function. Main factors of ferroptosis, lipid metabolism, ROS biology, and iron regulation, are also critically involved in developmental progress but hard to manipulate and investigate effectively in vivo through a high throughput way. Here in our study, an ex vivo culture model of tooth germ is applied to investigate the possible role of ferroptosis in organogenesis, since both RCD and risk factors of ferroptosis (lipid metabolism, ROS biology, and iron regulation) participate in tooth morphogenesis and the well-established ex vivo culture model enables effective manipulation of ferroptosis. To investigate whether ferroptotic stress exists and may influence tooth germ development, we detect the spatiotemporal expression of Gpx4, a core anti-lipid peroxidation enzyme in ferroptosis, in mouse development first molar and incisor. Morphological changes in each group with/without activation of ferroptosis are identified by gross anatomy, sequential HE staining, and related 3D reconstruction. Ferroptosis-related genes, lipid peroxidation, the morphology of mitochondria, and iron accumulation are detected to confirm the activation of ferroptosis. Inhibition of ferroptosis by DFO, Fer-1, and Lip-1, could partly rescue organogenesis of erastin-impaired tooth morphogenesis. Moreover, apoptosis is not significantly activated in erastin-impaired tooth germ indicated by cleavage of Caspase-3 and TUNEL staining. These results demonstrate that ferroptotic stress exists in tooth organogenesis but is well controlled by following upregulation of Gpx4; Overburdened ferroptotic stress cause by erastin significantly suppress tooth morphogenesis.