Pena de Castro, S.C.; (2011) Proteomic and molecular analysis of neural tube defects in the mouse embryo. Doctoral thesis , UCL (University College London).

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Abstract

The aim of this project was to investigate the causes of spinal neural tube defects (NTDs), using the curly tail (ct/ct) mouse as a model system. The ct mutant allele corresponds to a hypomorphic allele of grainyhead-like-3 (Grhl3) gene. A two-dimensional protein gel electrophoresis (2-DE) based approach was used to compare the proteome profile of ct/ct embryos with a genetically matched wild-type strain at the stage of spinal neural tube closure. This analysis revealed a series of proteins whose abundance or 2-DE gel migration are abnormal in ct/ct embryos. Detailed follow-up analysis was performed on one protein, lamin B1. Differential migration of lamin B1 on ct/ct compared with wild-type 2-DE gels was found to result from a sequence change in Lmnb1, resulting in the deletion of a glutamic acid (E) in a region of 9 glutamic acids in the wild-type protein. Lamin B1 in ct/ct therefore only has 8 glutamic acids in this part of the protein. Further analysis showed that the lamin B1 variants functionally differ. Genetic crosses were performed to generate sub-strains of ct/ct mice carrying different combinations of the Grhl3 mutation and lamin B1 variants. These studies support the hypothesis that Lmnb1 can modify the risk of NTDs in the ct/ct strain. Finally, while ct/ct NTDs result from diminished Grhl3 expression, the effects of Grhl3 over-expression were also investigated by intercrossing curly tail Grhl3-transgenic mice (ct/ctTgGrhl3). High levels of Grhl3 expression were found to cause NTDs at high frequency, indicating that Grhl3 regulation is an important requirement for neural tube closure. Morphological and gene expression analysis in Grhl3 over-expressing transgenic embryos suggest that the cellular mechanism underlying NTDs differs from that in the ct/ct hypomorphic mutant.

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