Developmental gene expression profile of Vmo1 in the mouse auditory system
Forrester-Gauntlett, B. K. E. (2013). Developmental gene expression profile of Vmo1 in the mouse auditory system (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/8449
Permanent Research Commons link: http://hdl.handle.net/10289/8449
Hearing loss (HL) is a sensory disorder that affects an estimated 250 million people worldwide and can greatly affect quality of life. In New Zealand, more than 10% of the population is affected by HL with the Māori population being overrepresented among all age groups. Therefore, understanding the mechanism of HL is extremely important for the development of new pharmaceuticals for the prevention or treatment of HL disorders. The main aim of the research undertaken in this thesis was to characterise the function of the Mus musculus (mouse) vitelline membrane outer layer one (Vmo1) gene. This gene is considered an excellent candidate for being involved in human HL and/or balance disorders. Our hypothesis is based on its restricted gene localisation within the mouse inner ear and the postulated function of Reissner’s membrane. Two methods were used to address this aim. Firstly, comparative genomics was used to determine the level of nucleotide and amino acid conservation of VMO1 across mammalian species, and to search for DNA motifs that may imply a biological function. Secondly, molecular biology and histochemical techniques were used to DNA sequence the Vmo1 gene, detect the expression of 22 kDa VMO1 protein within mouse tissues, and to localise the expression of VMO1 protein within the mouse inner ear. Comparative genomics results showed VMO1 to be highly conserved across 36 species. An in-depth analysis of the differences and similiarites between the mouse, human and chicken indicated a high level of gene conservation with an even greater degree of identity and similarity seen at a proteomic level. In addition, a high level of conservation across amino acids involved in the formation and stabilisation of the three dimensional structure. Thus, results suggest an important function for the VMO1 protein. Two commerical VMO1 antibodies were purchased to determine the localisation of the mouse VMO1 protein. They were validated for specificity using western blot analysis of protein lysates dissected from postnatal day 28 mice (P28). VMO1 was identified within the inner ear protein lysate and tear gland protein lysate of an expected molecular weight size of 20-37kDa with additional binding observed in the ear sample at 250kDa. Immunohistochemistry detected high concentrations of VMO1 protein within the tectorial membrane (TM) and inner pillar cells (IPC) in inner ear sections from the mouse at P5. In agreement with the comparative genomics analysis, VMO1 is a secreted protein. The movement of the hair cells (HC) relative to the TM is is essential for the transduction of sound into electrical signals. The IPC act as supporting cells for the hair cells, and help to couple movement of the basilar membrane to the HC. In conclusion, the importance of the TM and IPC in hearing function, and the localisation of the VMO1 protein within these structures implies an important role for VMO1 in hearing function. We recommend further studies to examine the specificity of the VMO1 antibody, and the development of a Vmo1 knockout mouse to support the functional analysis of Vmo1 in the auditory system.
University of Waikato
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