Molecular determination of novel genes and pathways required for vestibular morphogenesis in zebrafish

The vestibular system of the vertebrate inner ear functions to detect gravity and motion in order to maintain balance. Functionally there are two divisions of the vestibule: the semicircular canals which detect angular acceleration and the otolith organs which perceive linear movements. Each division, although structurally distinct, detects movement and gravity though the deflection of mechanosensory hair cells.;The goal of my research is to use zebrafish as a tool for the identification and characterization of genes that participate in vestibular development. I have discovered several genes that are expressed in the zebrafish otic vesicle during embryogenesis, and I have shown that these genes are essential for the formation of vestibular structures.;The first genes that I analyzed were the two zebrafish orthologs of Pten, a well studied tumor suppressor gene that is involved in many human cancers. This gene is known to control cell growth and proliferation through its negative regulation of the Akt pathway. The zebrafish orthologs, ptena and ptenb are expressed during embryonic development, and their gene products are able to regulate the levels of phorphorylated Akt in embryogenesis. The two genes show distinct expression patterns, and predictably, knockdown of the individual genes also produce distinct phenotypes. ptena, but not ptenb, is expressed in the otic vesicle. Knockdown of ptena produces otolith defects at 24 hpf (hours post fertilization) and semicircular canal defects at 48 and 72 hpf. Knockdown of ptenb does not affect inner ear development. These results suggest that the function of one ancestral Pten may have been divided between two orthologs after the genome duplication in teleost fish. I also conclude that control of cell growth and survival may play a role in the formation of these vestibular structures.;I have identified and investigated a novel zebrafish ortholog of a mammalian gene involved in otolith development. The mammalian otolith organs contains thousands of minute biomineralized particles called otoconia, whereas the inner ear of teleost fish contains three large ear stones called otoliths that serve a similar function. Otoconia and otoliths are composed of calcium carbonate crystals condensed on a core protein lattice. Otoconin-90 (Oc90) is the major matrix protein of mammalian and avian otoconia, while Otolith Matrix Protein-1 (Omp-1) is the most abundant matrix protein found in the otoliths of teleost fish. This difference in major matrix protein composition has been hypothesized to account for the morphological differences observed between mammalian otoconia and zebrafish otoliths. Therefore, it was unexpected that orthologs for these matrix proteins would be found other species. I have identified a novel gene, otoc1, which encodes the zebrafish ortholog of Oc90. Expression of otoc1 is detected in the ear between 15 hpf and 72 hpf, and is restricted primarily to the macula. During embryogenesis, expression of otoc1 mRNA precedes the appearance of omp-1 transcripts. Knockdown of otoc1 mRNA translation with antisense morpholinos produces a variety of aberrant otolith phenotypes. My results suggest that Oc90 orthologs may serve to nucleate calcium carbonate mineralization of zebrafish otoliths, and that this protein is not strictly involved in determining mammalian otoconial morphology.;The semicircular canals are three fluid filled tubes arranged in different orthogonal planes. Angular rotation of the head creates uneven flow of fluid through the canals and over the sensory hair cells that lie at each terminal, which can be translated to the brain via the eighth cranial nerve. The otolith organs, including the utricle and the saccule, are each composed of a patch of sensory hair cells associated with an ear stone composed of calcium carbonate.;Previous experiments have shown that a zebrafish ortholog of Neuronal Calcium Sensor-1 (Ncs-1) is required for the formation of semicircular canal hubs in zebrafish otogenesis (Blasiole et al. 2005). In order to gain further insight into the pathways involved in this Ncs-1 dependent process, I studied the role of Ncs-1 interacting proteins (NIPs) in vestibular development. A yeast-2-hybrid screen was performed to identify novel NIPs. Several of these newly identified and other previously known interactors were analyzed as candidates for otogenic genes. I determined that many zebrafish NIP orthologs are expressed in the developing semicircular canal structure. Morpholino knockdown of three of these genes, arf1, pi4kbeta and dan has demonstrated that these genes are indeed important for vestibular morphogenesis. Combinatorial knockdowns have also been used to show that arf1, pi4kbeta, and ncs-1a functionally interact. These functional interactions and direct physical associations suggest that these genes are involved in a unified pathway during inner ear development. (Abstract shortened by UMI.)...