Molecular studies of neural tube defect development in the mouse embryo

The thesis describes studies of the development of neural tube defects (NTD) in genetically predisposed mice, specifically the curly tail and loop-tail mutants. The curly tail mouse exhibits delay or failure of closure of the neural tube at the posterior neuropore and is a model for low spinal NTD in humans. In vivo supplementation of embryos with the vitamin, inositol, significantly reduces the incidence of spinal defects raising the possibility of using inositol to prevent a proportion of NTD in humans. Moreover, inositol treatment of embryos in culture minimises the delay in posterior neuropore closure that is known, from previous studies, to lead directly to development of NTD. The mechanism of action of inositol has been examined using in vitro inhibitors and activators, measurements of inositol incorporation and analysis of gene expression in cultured embryos. The findings suggest a model that involves an increased flux through the inositol/lipid cycle which leads to activation of protein kinase C and upregulation of expression of retinoic acid receptor-P in the hindgut, the affected tissue in the curly tail mutant. The loop-tail mutant mouse is a model for craniorachischisis in humans; homozygous embryos exhibit failure of initial closure of the neural folds resulting in an open neural tube from the midbrain/hindbrain boundary along the entire body axis. Loop-tail embryos are identified by PCR analysis prior to the failure of neural tube closure. Analysis of gene expression by whole mount in situ hybridisation reveals abnormal expression of sonic hedgehog and netrin-1 in the notochord and floor plate of homozygous mutant embryos suggesting that defects in the development of these tissues may contribute to the development of NTD. ANOVA Analysis of Variance AP-2 Activator protein-2 BCA Bicinchoninic acid BCIP 5-bromo-4-chloro-3-indolyl-phosphate bp Base pairs cAMP Cyclic adenosine monophosphate cDNA Complementary DNA cGMP Cyclic guanosine monophosphate ConA Concanavalin A CRABP Cellular retinoic acid binding protein CRBP Cellular retinoid binding protein DAG sn-1,2-diacylglycerol DEPC Diethyl pyrocarbonate DIG Digoxygenin DLHP Dorsolateral hinge points DNA Deoxyribonucleic acid DPH Diphenylhydantoin dpm Disintegrations per minute GPI Glycosylphosphatidylinositol EDTA Ethylene-diamine tetra-acetic acid HNF Hepatocyte nuclear factor IP3 Inositol triphosphate MARCKS Myristoylated alanine-rich C-kinase substrate MEM Modified Eagles Medium MHP Median hinge point MRP MARCKS-related protein NBT Nitroblue tétrazolium chloride NCAM Neural cell adhesion molecule NTD Neural tube defects OD Optical density PAX Paired box PBS Phosphate buffered saline PBT PBS, 0.1% Tween-20 PCR Polymerase chain reaction PFA Paraformaldehyde PG Prostaglandin PI Phosphatidylinositol PIP2 Phosphatidylinositol 4,5-biphosphate PKC Protein kinase C PNP Posterior neuropore RA Retinoic acid RAR Retinoic acid receptor Rf Relative front value RNA Ribonucleic acid SDS Sodium lauryl sulphate SEM Standard error of the mean Shh Sonic hedgehog SSC Sodium chloride/ sodium citrate buffer TBE Tris-borate EDTA electrophoresis buffer TE Tris-EDTA TEC Thin-layer chromatography TPA 12-0-tetradecanoyl-phorbol-13-acetate tRNA Transfer RNA UV Ultra violet ZPA Zone of polarising activity