Medical Student McGovern Medical School at the University of Texas Health Science Center at Houston Lantana, Texas, United States
Background: Our recent collaborative work has identified human mutations correlated with neural tube defects (NTD). These studies compiling 511 Whole Exome Sequences from Mexican American and European American myelomeningocele (MMC) patients have identified 18 genes with deleterious variants associated with cell migration and central nervous system (CNS) development, including the focal adhesion kinase (FAK) gene also known as protein tyrosine kinase 2 (PTK2). This gene encodes a non-receptor protein tyrosine kinase with significant roles in CNS development. Seven deleterious FAK variants associated with MMC were identified. Our preliminary cultured mouse embryonic fibroblast (MEF) data indicate that all seven patient FAK protein variants expressed significantly reduced cell migration. Objective: This project aims to evaluate the effects of two FAK variants (E900K and S940G), missense mutations within the focal adhesion targeting (FAT) domain, in NTD in Xenopus. These FAK variants resulted in the most retarded migration of MEF cells in culture. Previously, Fak knockdown in Xenopus showed NTD. In this study, we aimed to knock down endogenous Fak, evaluate NTD in Xenopus, and rescue phenotypes using the human wildtype (wt) FAK and FAK with patient variants (E900K and S940G). Design/Methods: 20ng and 30ng of Fak morpholinos (MO) were injected at 1-2 cell stages of Xenopus embryos. A standard MO was injected at the same concentration for control. Phenotypes of the developing embryos were compared at the end of gastrulation and neurulation using a stereomicroscope. Western blot analysis was used to confirm knockdown of the endogenous Fak and expression of the exogenous human FAK in the Xenopus embryo. In our preliminary data, 30ng Fak MO injections produced more significant gastrulation and NTD. phenotypes than 20ng injections. Western blots confirmed the reduction of endogenous Fak. We confirmed the expression of exogenous human wt and variant FAK protein in Xenopus embryos using Western blots. Overexpression and rescue of knockdown embryos with human FAK wt and variants are underway.
