Date of Award

12-2013

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Kenneth Johnson

Second Committee Member

Gregory Cox

Third Committee Member

Clarissa Henry

Abstract

The mammalian inner ear is a complex structure that requires specific events for proper formation to occur. Here I describe a previously unreported spontaneous mouse mutation named deaf wanderer (dwnd) that results in a shortened cochlea, supernumerary inner and outer cochlear hair cells, moderate hearing loss, and some balance defects. Using a positional cloning approach, we were able to show that this mutation is a 53 kb deletion in the multiple C2 and transmembrane protein 1 gene (Mctp1), a gene whose function has not been determined.

In order to identify a possible gene function, we investigated whether Mctpl could be acting in the Notch signaling pathway since other mouse mutants of Notch pathway members exhibit a similar phenotype. Using in situ hybridization to examine gene expression for different intermediates in the Notch pathway, we looked for differences in expression between mutant and control animals to identify a possible role for Mctpl in the Notch signaling pathway. We were unable to find any differences in gene expression between mutant and control animals for any of the genes tested, indicating that Mctp1 is probably not involved in the Notch pathway.

Mutations in genes essential for cell cycle exit also result in extra rows of hair cells. We used BrdU and PCNA immunostaining to investigate a possible failure of cochlear hair cells from dwnd animals to exit the cell cycle at the correct embryonic stage. We did not observe any staining in the hair cells of dwnd mutant animals after the period when cell cycle exit should have occurred, indicating that hair cells from these animals exit the cell cycle at the correct time.

A third cause of extra rows of cochlear hair cells are defects in the elongation process of the cochlea known as convergent extension. Cochlear measurements showed that the dwnd mutant cochlea is 23% shorter than that of its littermate control. We also found that while the inner and outer hair cell densities are greater in certain regions of the mutant than in the control, the total number of hair cells is significantly greater in the mutant than in the control. Convergent extension is a calcium dependent process, and the major function of the C2 domains in the MCTP1 protein is to bind calcium. It appears most likely that the phenotype of the dwnd mouse is due to a defect in the convergent extension process.

Download

Share