Cellular and Molecular Analysis of the Role of Inositol in Neurulation

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DESCRIPTION (provided by applicant): Neural tube defects (NTDs) are among the most frequent birth defects in humans, with a frequency of 1/1000 for spinal bifida alone. Given the high frequency of NTDs, an important area of research focus has been on preventative methods. In many cases NTDs can be prevented by maternal supplementation with folic acid. Nevertheless, around 30% of NTDs are unresponsive to this dietary supplement and there is currently no therapy available for these defects. Increasing evidence suggests that inositol supplementation can help prevent folate-resistant NTDs and a clinical trial is currently underway to test this possibility. Our long-term objective is to understand how inositol exerts its protectve effect during neurulation. The goal of this proposal is to investigate a putative mechanism by which inositol metabolism promotes neural convergence extension (CE), an early and essential stage of neurulation, using zebrafish as a model system. We will address the general hypothesis that Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), the kinase that generates inositol hexakisphosphate (IP6), interacts with the ciliary protein Ift88 to mediate the polarized cell movements that drive neural CE. In Aim 1 we will investigate the mechanism by which Ift88 functions in neural CE. Our working hypothesis is that Ift88 nucleates cytoplasmic microtubules (MTs), which we know to be required for polarized cell movement. This hypothesis will be tested by examining the integrity of the MT network in Ift88-depleted embryos. In Aim 2 we will determine if Ipk1 promotes neural CE by regulating MTs in an Ift88-dependent manner. This research goal will be achieved by comparing the phenotypes observed in Ipk1, Ift88 and Ipk1; Ift88 double mutants and testing the requirement for Ipk1 in the centrosome, the organelle that nucleates MTs. The proposed work should increase our understanding of the function of Ift88 and Ipk1, reveal essential mechanisms underlying neural tube formation and provide the basis for a model on the protective effect of inositol.