Aziz, N.M.A.; (2006) Molecular basis of neural fold adhesion and fusion in closure of the spinal neural tube. Doctoral thesis , University of London.

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Abstract

The molecular basis of neural fold adhesion and fusion in closure of the mouse spinal neural tube is a crucial process, since failure may result in spina bifida. Previous studies have shown that cleavage of glycosyl phosphatidylinositol (GPl)-anchored proteins causes neural tube defects in the mouse. In this study, mouse embryos undergoing neural tube closure were treated with phosphatidylinositol specific phospholipase C (PIPLC) enzyme known to cleave GPI-anchored molecules, and then cultured for 8 hours. PIPLC treatment inhibits spinal neural tube closure, as shown by an enlarged posterior neuropore. EphrinAs are GPI-anchored cell surface proteins, and were considered as candidates for a role in adhesion. Perturbation of ephrinA ligand binding to EphA receptors by injecting EphA3 fusion protein into the amniotic sacs of cultured embryos, inhibits neural tube closure. Further microinjection experiments with EphAl fusion protein, which also inhibits closure, suggests that the specific ephrinA ligand, ephrinA 1, is required for spinal neurulation. Expression studies show localization of ephrinA 1, ephrinA3 and the EphA2 receptor in the spinal neural tube. Further work has involved RT-PCR to investigate the expression of possible ephrin binding partners and has shown that EphAl, EphA3, EphA4, EphA5, EphB2, EphB3, EphB4 and EphB6 are also expressed in the spinal neural tube. The ephrinA 1 ligand is currently being studied further by constructing a conditional knockout murine model of the gene. The EphA2 receptor shows an interesting pattern of expression in the spinal neuroepithelium and surface ectoderm during neurulation. The EphA2 receptor is expressed specifically at the point of closure and displays fluctuating amounts of expression on the tips of apposing neural folds. The fluctuating mRNA expression of EphA2 in apposing tips of neural folds may indicate a genetic role of an asymmetrical protrusion from the tip of the left neural fold as viewed via electron microscopy. EphA2 is expressed on the lamellipodia-like protrusion which emanates from the left neural fold. The findings strongly suggest that the Eph/ephrin system plays a role in determining the structure of the initial attractive properties of the tips of the apposing neural folds during adhesion and fusion of the spinal neural tube which culminates in the appearance of an apoptotic cell at the front line of closure.

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