A critical period of ear development controlled by distinct populations of ciliated cells in the zebrafish

The zebrafish (Danio rerio) is a useful model system for analyzing development of the inner ear. A number of mutations affecting the inner ear have been identified. Here we investigate the initial stages of otolith morphogenesis in wild-type embryos as well as in monolith (mnl) mutant embryos, which fail to form anterior otoliths but otherwise appear normal. Otolith growth is initiated at 18-18.5 h by localized accretion of free-moving precursor particles. This process, referred to as otolith seeding, is regulated by two classes of cilia: First, kinocilia of precociously forming hair cells (tether cells) bind seeding particles, thereby localizing otolith formation. Tether cells usually occur in pairs at the anterior and posterior ends of the ear. Despite the presence of functional kinocilia, tether cells initially appear immature and do not acquire the characteristics of mature hair cells until approximately 21.5 h. Second, beating cilia distributed throughout the ear agitate seeding particles, thereby inhibiting premature agglutination. Constraining particles with laser tweezers caused them to fuse into large untethered masses. Bringing such masses into contact with tethered otoliths caused them to fuse, greatly enhancing otolith growth. Selectively enhancing one otolith greatly inhibited growth of the second, creating an imbalance that persisted for many days. Seeding particles and beating cilia disappear soon after 24 h, and the rate of otolith growth decreases by nearly 90%. In mnl mutant embryos, tethers and beating cilia are distributed normally, but anterior otoliths fail to form in 80-85% of mutant ears. The binding properties of seeding particles appear normal, as shown by their ability to fuse when entrapped by laser tweezers and their binding to posterior tethers. We infer that anterior tethers have a weakened ability to bind seeding particles in mnl embryos. Immobilizing mnl embryos with the anterior end of the ear oriented downward effectively concentrated the dense seeding particles near the anterior tethers and permitted all to form anterior otoliths. However, immobilizing mnl embryos after 24 h when seeding particles were depleted did not facilitate anterior otolith formation. Together, these data demonstrate that the ability to initiate otolith formation is limited to a critical period, from 18.5 to 24 h, and that interfering with the functions of tether cell kinocilia or beating cilia impairs otolith seeding and subsequent otolith morphogenesis.