Molecular and Functional Analysis of Otolith Formation in Zebrafish, Danio Rerio

148 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.Mammalian otoconia and fish otoliths are calcium-carbonate biominerals and important inner-ear organs that detect linear accelerations and perceive gravity. As a result, the absence, degradation, or dislocation of these structures causes severe imbalance in affected animals. The common vestibular defect called "benign paroxysmal positional vertigo" (BPPV) in humans results from degradation of otoconia, which causes severe dizziness. Therefore, BPPV increases the risk of falls and lead to injuries or death in the elderly. Unfortunately, the clinical treatments for BPPV are limited, and BPPV often recurs in treated patients. In order to understand the molecular mechanisms of otolith and otoconia biomineralization, we performed a biochemical analysis of fish otoliths. Although there are more than two-dozen proteins in otoliths, only few of them are identified and known to be involved in otolith formation. Therefore, the goal of Chapter 2 is to identify unknown otolith genes by proteomics and investigate their roles in otolith formation. As a result, we identified three novel otolith-matrix proteins: Precerebellin-like protein (Cbinl), Neuroserpin (SerpinI1), and Sparc. A loss-of-function study revealed that sparc is required for normal otolith morphogenesis. In Chapter 3, in order to reveal the mechanisms that control otolith formation, we established a reliable centrifuge setup that perturbs normal otolith formation by raising fertilized eggs at simulated hypergravity for 8 days. Immediately after centrifugation, fewer fish survived, otolith sizes were significantly reduced, and the probability of losing an otolith increased with linear acceleration compared to fish at 1 x g control. When fish embryos were raised at low linear accelerations from 3 to 6 x g, where general side effects are absent, the mRNA levels of oc90 and otol1a were significantly down-regulated compared to those of controls. In conclusion, the novel discovery of three additional otolith-matrix proteins in Chapter 2 has expanded our molecular understanding of otolith formation. Future studies will be able to utilize centrifugation to reveal the molecular pathways that regulate the ions and proteins critical for otolith formation.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD