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.

This paper presents a frequency-domain ultrasonic technique for a simultaneous determination of the thickness (h) and wave speed (c) of the individual layers comprising a multilayered medium. The layers may be "thin"; by thin we mean that the successive reflections of an ultrasonic pulse from the two faces of a layer are nonseparable in the time domain. Plane longitudinal waves which are normally incident upon the medium are considered. A systematic analysis of the sensitivity of the complex-valued transfer function to the acoustical parameters of each layer has been carded out. An inverse algorithm, which utilizes either the NewtonRaphson or the Simplex method in conjunction with the incremental search method, has been developed to reconstruct simultaneously the thickness and phase velocity of each layer by minimizing the difference between the theoretical and the experimental results in the mean-sum-square sense; the entire complex spectrum, i.e., the amplitude as well as the phase spectrum, was used. The technique is fully automated and computer controlled and can be readily used for in situ NDE applications. Results are presented for several three-layer specimens; aluminum/water/aluminum, aluminum/water/titanium, and titanium/water/ titanium. Successful inversion was obtained for the following cases (1) simultaneous determination of h and c of any one of the three layers, given h and c of the remaining two layers;(2) simultaneous measurement of the three thicknesses, given the three wave speeds; (3) simultaneous measurement of the three wave speeds, given the three thicknesses; (4) simultaneou• determination of all three thicknesses and one wave speed, given the remaining two wave soet.ls. The precision of our measurements was found to be excellent; typically, q-3 pm in h (for h of the order of 1 mm) and q-one part per thousand in c. The accuracy was found to be about one order of magnitude lower than the precision; typically, q-10 pm in h and q-2% in c.

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