Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

January 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

David Ornitz

Abstract

Otopetrin family genes encode multi-transmembrane domain proteins with three highly conserved domains. In mice, three Otopetrin paralogues are found. One of its members, Otopetrin 1: Otop1) has been previously shown to be essential for the formation of otoconia in the vestibular system of the inner ear. Otoconia are calcium carbonate biominerals that are required for normal balance and the sensation of linear acceleration with respect to gravity. The mechanism by which OTOP1 mediates otoconia biosynthesis is not known, but the ability of OTOP1 to modulate [Ca2+]i in response to purinergic signals in heterologous systems suggest that OTOP1 may be involved in concentrating [Ca2+] within the sensory epithelium and/or the globular substance vesicles, which are secreted from the epithelium. In this study, we generated null alleles of Otop1: Otop1βgal/βgal) and Otop2: Otop2nβgal/nβgal) in mice to investigate the in vivo functions of these genes. The otoconial agenesis phenotype in Otop1βgal/βgal mice suggests that the most sensitive and important role for OTOP1 during development is mediating otoconia formation. X-gal staining and immunohistochemical analysis reveal that Otop1 is expressed in the developing and adult supporting cells in the non-striola region of the maculae. Within these sensory epithelia, OTOP1 localizes near the apical end. In primary utricular macular cultures, endogenous OTOP1 is necessary to inhibit P2Y function and the influx of extracellular Ca2+ in a Ca2+- and ATP-dependent manner. The two different recessive missense mutations of Otop1, tilted: tlt) and mergulhador: mlh), result in the same phenotype as in Otop1βgal/βgal, suggesting that they have inactivated essential OTOP1 activity. Localization studies and ratiometric Ca2+ imaging in COS7 cells and primary utricular macular cultures show that both tlt and mlh alter normal localization of OTOP1 and that the mlh mutation has a greater effect on modulating [Ca2+]i compared to tlt. When examined in heterologous systems, OTOP2 and 3 can modulate [Ca2+]i in a similar manner to OTOP1, suggesting possible functional redundancy between Otopetrins. However, the double knockout allele for Otop1 and Otop2 does not exhibit additional phenotypes, suggesting that these genes may have more prominent functional redundancy with OTOP3. Alternatively, a subtler phenotype may exist but will require a more careful analysis to be revealed. Because otoconia formation is affected by altered gravity it is possible that the genes important for otoconia biosynthesis, such as Otoconin90 and Otop1, may also be affected. However, a quantitative RT-PCR analysis of quail embryo development under constant hypergravity conditions shows that expression of these genes is not significantly affected by altered gravity treatment. This result indicates that gravity is unlikely to serve as an epigenetic factor for these two genes during development of otoconia.

Comments

Permanent URL: http://dx.doi.org/10.7936/K70V89TC

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