Neural tube defects (NTDs) are common birth defects in humans. Although it is known that genetic susceptibility factors are associated with NTDs in humans, they have yet to be identified. The mouse can be used as a model system to study genetic factors associated with NTDs and the mechanism of neural tube closure. In the course of studying mice with inactivation of each of the three mouse Disheveled (Dvl) genes, we discovered that Dvl2 -/- and Dvl1 -/- Dvl2 -/- display NTDs. Disheveled is an important gene in the evolutionarily conserved Wnt/wingless signal transduction pathway and the planar cell polarity (PCP) pathways. All eukaryotic Dvl proteins contain three highly conserved domains: DIX, PDZ and DEP. Studies in Drosophila and Xenopus indicate that while the DIX and N-terminal portion of the DEP domain are required for Wnt pathway signaling, the PDZ and C-terminal DEP domain are essential for the PCP/convergent extension pathway. Disruption of either the Wnt or PCP pathways may result in NTDs. We propose to determine the spatial and temporal requirements for Dvl proteins during neural tube closure, and whether the NTDs displayed by Dvl mutant mice are caused by Writ signaling defects and/or PCP pathway defects. We will use the following specific aims. 1) To further characterize Dvl-dependent pathways responsible for the NTDs in Dvl 1/2 mutants, we will examine Wnt and PCP/convergent extension pathway signaling in cells and in vivo during neural tube closure in wild-type and Dvl 1/2 double mutants. 2) We will test for genetic interactions between Loop tail/strabismus (Stbm) and fused/axin with Dvl1 and Dvl2 in neural tube closure, to genetically distinguish PCP and Wnt pathway effects, respectively. 3) To determine the sites in neural tube that require Dvl2 function for normal closure, we will produce mice with specific loss-of-function of Dvl2 or specific expression of Dvl2 in spatially and temporally restricted patterns. The effect of these mutations on neural tube closure in wild type and Dvl mutants will be assessed. 4) To determine the domains of the Dvl2 protein that are required for neural tube closure, we will produce an allele series of Dvl2 mutants in mice using a BAC transgenic strategy. Precise mutations in Dvl2 will be engineered using homologous recombination of BACs in bacteria, and the resultant mutant Dvl2 BACs will be used to produce transgenic mice. The effect of these mutations on neural tube closure in Dvl -/-Dvl2 -/- mutants will be assessed.
