Characterisation and functional analysis of a novel msp domain – containing protein, MOSPD1
MOSPD1 belongs to a class of proteins that have a major sperm protein (MSP) domain at the N terminus and two transmembrane domains at the C terminus and are thought to act as membrane adaptor proteins. Previous work in the laboratory indicated that the closely related, mammalian-specific, Mospd 3 plays a role in the development and function of the heart as homozygous Mospd 3 gene trap neonates displayed a right ventricle defect characterised by a thinning of the right ventricle wall. The function of Mospd 1 is not known. Whilst Mospd 3 is mammalian specific Mospd 1 is conserved in all vertebrates including Danio rerio (zebrafish). The aims of this thesis were to investigate the possibility of genetic redundancy between Mospd 1 and Mospd 3 by identifying the sub-cellular localisation of MOSPD1 and MOSPD3 in both cells and tissues and to investigate the function of MOSPD1. Mouse monoclonal antibodies specific for MOSPD1 and MOSPD3 were generated and tested to ensure they did not cross react. MOSPD1 was found to be localised to the nucleus whilst MOSPD3 was located in the nucleus and cytoplasm. The sub-cellular localisation of these proteins changes during the cell cycle as they were localised to the cytoplasm during cell division, possible due to the breakdown of the nuclear membrane during cell division. To investigate the function of Mospd 1 during early development Mospd 1 gene expression was knocked down using morpholino anti-sense knockdown technology. A morpholino was generated against the splice-site between exons 2 and 3 of the zebrafish Mospd 1 gene and injected into early embryos. At doses that significantly reduced the level of Mospd 1, to below 50 %, the embryos developed normally and did not exhibit any gross morphological phenotypes when compared to both noninjected and 5 mispair control morpholino-injected embryos. A morpholino targeted to the start site of the Mospd 1 gene confirmed the lack of a gross morphological phenotype. In conjunction with the zebrafish functional tests the tools were generated to assess the role of Mospd 1 in a mammalian system. A conditional allele of Mospd 1 was generated in mouse embryonic stem (ES) cells which could be used to generate a conditional Mospd 1 mouse. The electroporation of a Cre recombinase plasmid into the conditional Mospd 1 ES cell line resulted in the generation of Mospd 1 null ES clones which could be used for functional studies both in vitro and in vivo. The Mospd 1 null ES cells were able to self-renew, expressed ES cell specific markers and were able to differentiate into cardiomyocytes. However, Mospd 1 null cells showed a reduced ability to differentiate into osteoblasts compared to wild type cells and showed changes in the expression of genes involved in Epithelial to mesenchymal transition (EMT) indicating Mospd 1 may be involved in this process.